Human tumor necrosis factor tr21 and methods based thereon

ABSTRACT

The present invention relates to TR21 polypeptides. In particular, isolated nucleic acid molecules are provided encoding human TR21 protein. TR21 polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of TR21 activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a divisional of U.S. application Ser. No.12/506,060, filed Jul. 20, 2009, which is a continuation of U.S.application Ser. No. 11/517,438, filed Sep. 8, 2006, which is adivisional of U.S. application Ser. No. 10/229,352, filed Aug. 28, 2002(now U.S. Pat. No. 7,112,410, issued Sep. 26, 2006), which claimsbenefit under 35 U.S.C. §119(e) of U.S. Provisional Application No.60/315,357 filed Aug. 29, 2001, each of which is hereby incorporated byreference in its entirety.

REFERENCE TO SEQUENCE LISTING AS TEXT FILE

This application refers to a “Sequence Listing” listed below, which isprovided as a text file. The text file contains a document entitled“PF562D1C1D1_SequenceListing.txt” (4,766 bytes, created Nov. 10, 2011),which is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to TR21, a member of the tumor necrosisfactor family of receptors. More specifically, isolated nucleic acidmolecules are provided encoding TR21 and variants thereof. TR21polypeptides are also provided, as are vectors, host cells, andrecombinant and synthetic methods for producing the same. The inventionalso relates to diagnostic and therapeutic methods using TR21 nucleicacid molecules, polypeptides and/or TR21 agonists or antagonists, suchas for example agonistic anti-TR21 antibodies, and antagonisticanti-TR21 antibodies. The invention further relates to screening methodsfor identifying agonists and antagonists of TR21 activity.

2. Related Art

Many biological actions, for instance, response to certain stimuli andnatural biological processes, are controlled by factors, such ascytokines. Many cytokines act through receptors by engaging the receptorand producing an intra-cellular response.

For example, tumor necrosis factors (TNF) alpha and beta are cytokines,which act through TNF receptors to regulate numerous biologicalprocesses, including protection against infection and induction of shockand inflammatory disease. The TNF molecules belong to the “TNF-ligand”superfamily, and act together with their receptors or counter-ligands,the “TNF-receptor” superfamily. So far, more than ten members of the TNFligand superfamily have been identified and more than ten members of theTNF-receptor superfamily have been characterized.

Among the ligands there are included TNF-alpha, lymphotoxin-alpha(LT-alpha, also known as TNF-beta), LT-beta (found in complexheterotrimer LT-2-beta), FasL, CD4L, CD27L, CD30L, 4-1BBL, OX40L andnerve growth factor (NGF). The superfamily of TNF receptors includes thep55TNF receptor, p75TNF receptor, TNF receptor-related protein, FASantigen or APO-1, CD40, CD27, CD30, 4-1BB, OX40, low affinity p75 andNGF-receptor (A. Meager, Biologicals 22:291-295 (1994)).

Many members of the TNF-ligand superfamily are expressed by activatedT-cells, implying that they are necessary for T-cell interactions withother cell types which underlie cell ontogeny and functions. (A. Meager,supra).

Considerable insight into the essential functions of several members ofthe TNF receptor family has been gained from the identification andcreation of mutants that abolish the expression of these proteins. Forexample, naturally occurring mutations in the FAS antigen and its ligandcause lymphoproliferative disease (R. Watanabe-Fukunaga et al., Nature356:314 (1992)), perhaps reflecting a failure of programmed cell death.Mutations of the CD40 ligand cause an X-linked immunodeficiency statecharacterized by high levels of immunoglobulin M and low levels ofimmunoglobulin G in plasma, indicating faulty T-cell-dependent B-cellactivation (R. C. Allen et al., Science 259:990 (1993)). Targetedmutations of the low affinity nerve growth factor receptor cause adisorder characterized by faulty sensory innervation of peripheralstructures (K. F. Lee et al., Cell 69:737 (1992)).

TNF alpha and LT-alpha are capable of binding to two TNF receptors (the55- and 75-kd TNF receptors). A large number of the biological effectselicited by TNF alpha and LT-alpha are mediated through their receptors,include hemorrhagic necrosis of transplanted tumors, cytotoxicity, arole in endotoxic shock, inflammation, immunoregulation, proliferationand anti-viral responses, as well as protection against the deleteriouseffects of ionizing radiation. TNF alpha and LT-alpha are involved inthe pathogenesis of a wide range of diseases, including endotoxic shock,cerebral malaria, tumors, autoimmune disease, AIDS and graft-hostrejection (B. Beutler and C. Von Huffel, Science 264:667-668 (1994)).Mutations in the p55 receptor cause increased susceptibility tomicrobial infection.

Moreover, a domain of about 80 amino acids near the C-terminus of TNFR1(p55) and Fas was reported as the “death domain,” which is responsiblefor transducing signals for programmed cell death (Tartaglia et al.,Cell 74:845 (1993)).

Apoptosis, or programmed cell death, is a physiologic process essentialto the normal development and homeostasis of multicellular organisms (H.Steller, Science 267:1445-1449 (1995)). Derangements of apoptosiscontribute to the pathogenesis of several human diseases includingcancer, neurodegenerative disorders, and acquired immune deficiencysyndrome (C. B. Thompson, Science 267:1456-1462 (1995)). Recently, muchattention has focused on the signal transduction and biological functionof two cell surface death receptors, Fas/APO-1 and TNFR-1 (J. L.Cleveland et al., Cell 81:479-482 (1995); A. Fraser et al., Cell85:781-784 (1996); S. Nagata et al., Science 267:1449-56 (1995)). Bothare members of the TNF receptor family, which also include TNFR-2, lowaffinity NGFR, CD40, and CD30, among others (C. A. Smith et al., Science248: 1019-23 (1990); M. Tewari et al., in Modular Texts in Molecular andCell Biology M. Purton, Heldin, Carl, Ed. (Chapman and Hall, London,1995). While family members are defined by the presence of cysteine-richrepeats in their extracellular domains, Fas/APO-1 and TNFR-1 also sharea region of intracellular homology, appropriately designated the “deathdomain,” which is distantly related to the Drosophila suicide gene,reaper (P. Golstein et al., Cell 81:185-6 (1995); K. White et al.,Science 264:677-83 (1994)). This shared death domain suggests that bothreceptors interact with a related set of signal transducing moleculesthat, until recently, remained unidentified. Activation of Fas/APO-1recruits the death domain-containing adapter molecule FADD/MORT1 (A. M.Chinnaiyan et al., Cell 81:505-512 (1995); M. P. Boldin et al., J. Biol.Chem. 270:7795-8 (1995); F. C. Kischkel et al., EMBO 14:5579-5588(1995)), which in turn binds and presumably activates FLICE/MACH1, amember of the ICE/CED-3 family of pro-apoptotic proteases (M. Muzio etal., Cell 85: 817-827 (1996); M. P. Boldin et al., Cell 85:803-815(1996)). While the central role of Fas/APO-1 is to trigger cell death,TNFR-1 can signal an array of diverse biological activities-many ofwhich stem from its ability to activate NF-kB (L. A. Tartaglia et al.,Immunol Today 13:151-153 (1992)). Accordingly, TNFR-1 recruits themultivalent adapter molecule TRADD, which like FADD, also contains adeath domain (H. Hsu et al., Cell 81:495-504 (1995); H. Hsu et al., Cell84:299-308 (1996)). Through its associations with a number of signalingmolecules including FADD, TRAF2, and RIP, TRADD can signal bothapoptosis and NF-kB activation (H. Hsu et al., Cell 84:299-308 (1996);H. Hsu et al., Immunity 4:387-396 (1996)).

Recently, Human Genome Sciences has demonstrated that the TNF ligandfamily member Neutrokine-alpha (International publication number WO98/18921) induces both in vitro and in vivo B cell proliferation. Blymphocytes are responsible for the production of immunoglobulins, themajor effector molecules of the humoral immune system. Immune systemrelated disorders associated with B cells include, for example,immunodeficiencies and autoimmune disease.

Accordingly, there is a need to provide cytokines similar to TNF thatare involved in pathological conditions. Such novel cytokines may beused to make novel antibodies or other antagonists that bind theseTNF-like cytokines for diagnosis and therapy of disorders related toTNF-like cytokines. More particularly, there is a need to provideNeutrokine-alpha binding proteins that may be involved in pathologicalconditions. Such novel Neutrokine-alpha binding proteins may be used,for example, as therapeutics to treat or prevent diseases, disorders orconditions associated with aberrant Neutrokine-alpha mediated activity.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding at least a portion of TR21. Thus,the present invention provides, for example, isolated nucleic acidmolecules comprising a polynucleotide encoding the TR21 receptor havingthe amino acid sequence shown in FIG. 1 (SEQ ID NO:2).

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells. The invention further provides forthe use of such recombinant vectors in the production of TR21polypeptides by recombinant techniques.

The invention further provides an isolated TR21 polypeptide having anamino acid sequence encoded by a polynucleotide described herein.

The present invention also provides diagnostic assays such asquantitative and diagnostic assays for detecting levels of TR21 protein.Thus, for instance, a diagnostic assay in accordance with the inventionfor detecting over-expression of TR21, or soluble form thereof, comparedto normal control tissue samples may be used to detect the presence oftumors.

Tumor Necrosis Factor (TNF) family ligands are known to be among themost pleiotropic cytokines, inducing a large number of cellularresponses, including cell proliferation, cytotoxicity, anti-viralactivity, immunoregulatory activities, hematopoiesis, and thetranscriptional regulation of several genes. Cellular responses toTNF-family ligands include not only normal physiological responses, butsuch responses may lead to diseases associated with dysregulation ofthese physiological responses, such as, for example, diseases associatedwith increased apoptosis or the inhibition of apoptosis.Apoptosis-programmed cell death is a physiological mechanism involved inthe deletion of peripheral T lymphocytes of the immune system, and itsdysregulation can lead to a number of different pathogenic processes.Diseases associated with increased cell survival, unregulated cellproliferation, or the inhibition of apoptosis, include cancers,autoimmune disorders, viral infections, inflammation, graft vs. hostdisease, acute graft rejection, and chronic graft rejection. Diseasesassociated with increased apoptosis include AIDS, neurodegenerativedisorders, myelodysplastic syndromes, ischemic injury, toxin-inducedliver disease, septic shock, cachexia, and anorexia.

Thus, the invention further provides a method comprising contactingcells which express the TR21 polypeptide with a candidate compound and aTNF-family ligand (e.g. Neutrokine-alpha (International PublicationNumber WO 97/33902)), and assaying for the inhibition of TR21 mediatedsignaling, and/or activation of transcription factors, such as, forexample, AP-1 and/or NF-kappaB, induced by, for example, a TNF-familyligand (e.g., Neutrokine-alpha) which involves administering to a cellwhich expresses the TR21 polypeptide (e.g., a B cell) an effectiveamount of a TR21 antagonist capable of decreasing TR21 mediatedsignaling.

The present invention is also directed to methods for enhancing TR21mediated signaling induced by a TNF-family ligand (e.g.,Neutrokine-alpha) which involves administering to a cell which expressesthe TR21 polypeptide (e.g., a B cell) an effective amount of a TR21agonist capable of increasing TR21 mediated signaling.

Whether any candidate “agonist” or “antagonist” of the present inventioncan enhance or inhibit TR21 mediated signaling can be determined usingor routinely modifying TNF-family ligand/receptor cellular responseassays known in the art, including, for example, those described in vonBulow et al. (Science 278:138-141 (1997)) and herein (see, e.g.,Examples 17 and 18). Thus, in a further embodiment, a screening methodis provided for determining whether a candidate agonist or antagonist iscapable of enhancing or decreasing TR21 mediated cellular response toTNF-ligand (e.g., Neutrokine-alpha). The method involves contactingcells expressing TR21 with the candidate compound (i.e., candidateagonist or antagonist compound) and TNF-ligand (e.g., Neutrokine-alpha),and measuring the TR21 mediated cellular response (e.g., activation oftranscription factors such as, for example, NF-AT, AP-1, and/orNF-kappaB), and comparing the cellular response to a standard cellularresponse. The standard cellular response being measured when contact ismade with the TNF-family ligand (e.g., Neutrokine-alpha) in absence ofthe candidate compound. An increased cellular response over the standardindicates that the candidate compound is an agonist of the ligand (e.g.,Neutrokine-alpha)/TR21 signaling pathway and a decreased cellularresponse compared to the standard indicates that the candidate compoundis an antagonist of the ligand (e.g., Neutrokine-alpha)/TR21 signalingpathway. By the invention, a cell expressing the TR21 polypeptide can becontacted with either an endogenous or exogenously administeredTNF-family ligand (e.g., Neutrokine-alpha.).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino acidsequence (SEQ ID NO:2) of TR21. Predicted amino acids from about 1 toabout 81 constitute the extracellular domain (SEQ ID NO:2); amino acidsfrom about 82 to about 101 constitute the transmembrane domain (SEQ IDNO:2); and amino acids from about 102 to about 184 constitute theintracellular domain (SEQ ID NO:2).

FIG. 2 shows an analysis of the TR21 amino acid sequence. Alpha, beta,turn and coil regions; hydrophilicity; amphipathic regions; flexibleregions; antigenic index and surface probability are shown. The regionswere determined by analyzing the amino acid sequence of FIG. 1 (SEQ IDNO:2) using the default parameters of the recited computer programs. Inthe “Antigenic Index—Jameson-Wolf” graph, amino acid residues 3 to 15,39 to 45, 51 to 53, 59 to 61, 104 to 112, 115 to 122, 136 to 138, 147 to153 and 178 to 182 in FIG. 1 (SEQ ID NO:2) correspond to highlyantigenic regions of the TR21 protein.

A tabular representation of the data summarized graphically in FIG. 2can be found in Table I. In Table I, the columns are labeled with theheadings “Res,” “Position,” and Roman numerals I-XIV. The columnheadings refer to the Following Features of the amino acid sequencepresented in FIG. 2 and Table I: “Res”: amino acid residue of SEQ IDNO:2 and FIG. 1; “Position”: position of the corresponding residuewithin SEQ ID NO:2 and FIG. 1; “I”: Alpha Regions—Garnier-Robson; “II”:Alpha Regions—Chou-Fasman; “III”: Beta Regions—Gamier-Robson; “IV”: BetaRegions—Chou-Fasman; “V”: Turn Regions—Garnier-Robson; “VI”: TurnRegions—Chou-Fasman; “VII”: Coil Regions—Garnier-Robson; “VIII”:Hydrophilicity Plot—Kyte-Doolittle; “IX”: HydrophobicityPlot—Hopp-Woods; “X”: Alpha Amphipathic Regions—Eisenberg; “XI”: BetaAmphipathic Regions—Eisenberg; “XII”: Flexible Regions—Karplus-Schulz;“XIII”: Antigenic Index—Jameson-Wolf; “XIV”: Surface ProbabilityPlot—Emini.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding TR21, such as, for example,polynucleotides having the nucleotide sequence shown in FIG. 1 (SEQ IDNO:1). The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a TR21 polypeptide having the aminoacid sequence shown in FIG. 1 (SEQ ID NO:2).

TR21 Nucleic Acid Molecules

The determined nucleotide sequence of TR21 (FIG. 1; SEQ ID NO:1)contains an open reading frame encoding a protein of about 184 aminoacid residues, with a deduced molecular weight of about 18.9 kDa. Theamino acid sequence of the predicted mature TR21 receptor is shown inSEQ ID NO:2 from amino acid residue about 1 to residue about 184.

As indicated, the present invention also provides the mature form(s) ofthe TR21 receptors of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal orsecretory leader sequence which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated. Most mammalian cells and even insect cellscleave secreted proteins with the same specificity. However, in somecases, cleavage of a secreted protein is not entirely uniform, whichresults in two or more mature species on the protein. Further, it haslong been known that the cleavage specificity of a secreted protein isultimately determined by the primary structure of the complete protein,that is, it is inherent in the amino acid sequence of the polypeptide.

The present invention provides a nucleotide sequence encoding the matureTR21 polypeptide having the amino acid sequence shown in FIG. 1. By themature TR21 protein having the amino acid sequence shown in FIG. 1 ismeant the mature form(s) of the TR21 receptor predicted by computeranalysis or produced by expression of the coding sequence shown in FIG.1 in a mammalian cell (e.g., COS cells, as described below). Asindicated below, the mature TR21 receptor having the amino acid sequenceencoded by the coding sequence shown in FIG. 1 may or may not differfrom the predicted mature TR21 protein shown in FIG. 1 (amino acids fromabout 1 to about 184) depending on the accuracy of the predictedcleavage site based on computer analysis.

Methods for predicting whether a protein has a secretory leader as wellas the cleavage point for that leader sequence are available. Forinstance, the method of McGeoch (Virus Res. 3:271-286 (1985)) and vonHeinje (Nucleic Acids Res. 14:4683-4690 (1986)) can be used. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75-80% (vonHeinje, supra). However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

The polypeptide sequence of the TR21 depicted in FIG. 1 can routinely beexamined by computer programs. For example, the mature form,intracellular form, extracellular form, and transmembrane domains of theTR21 polypeptides of the invention can routinely be predicted viaanalysis using the “PSORT” computer program (K. Nakai and M. Kanehisa,Genomics 14:897-911 (1992)), which is an expert system for predictingthe cellular location of a protein based on the amino acid sequence. Aspart of this computational prediction of localization, the methods ofMcGeoch and von Heinje are incorporated into the PSORT program.

The predicted TR21 polypeptide comprises about 184 amino acids. However,as one of ordinary skill in the art would appreciate, the actual TR21polypeptide may be anywhere in the range of 174-194 amino acids due tothe possibilities of sequencing errors as well as the variability ofcleavage sites for leaders in different known proteins. It will furtherbe appreciated that, the domains described herein have been predicted bycomputer analysis, and accordingly, that depending on the analyticalcriteria used for identifying various functional domains, the exact“address” of, for example, the extracellular domain, intracellulardomain, cysteine-rich motif, and transmembrane domain of TR21 may differslightly from the predicted locations. For example, the exact locationof the TR21 extracellular domain in FIG. 1 (SEQ ID NO:2) may varyslightly (e.g., the address may “shift” by about 1 to about 20 residues,more likely about 1 to about 5 residues) depending on the criteria usedto define the domain. In any event, as discussed in more detail below,the invention further provides polypeptides having various residuesdeleted from the N-terminus and/or C-terminus of the complete TR21polypeptide, including polypeptides lacking one or more amino acids fromthe N-termini of the TR21 extracellular domains described herein, whichconstitute soluble forms of the extracellular domain of the TR21polypeptides respectively.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA may be the coding strand, also known as the sensestrand, or it may be the non-coding strand, also referred to as theanti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced naturally, recombinantly or synthetically. However, anucleic acid molecule contained in a clone that is a member of a mixedclone library (e.g., a genomic or cDNA library) and that has not beenisolated from other clones of the library (e.g., in the form of ahomogeneous solution containing the clone without other members of thelibrary) or a chromosome isolated or removed from a cell or a celllysate (e.g., a “chromosome spread”, as in a karyotype), or apreparation of randomly sheared or genomic DNA cut with one or morerestriction enzymes, is not “isolated” for the purposes of thisinvention.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) shown in FIG. 1 (SEQ IDNO:1); DNA molecules comprising the coding sequence for the complete(full-length) and/or mature TR21 protein shown in FIG. 1 (SEQ ID NO:2);and DNA molecules which comprise a sequence substantially different fromthose described above, but which, due to the degeneracy of the geneticcode, still encode the TR21 protein. Of course, the genetic code is wellknown in the art. Thus, it would be routine for one skilled in the artto generate such degenerate variants.

The invention further provides an isolated nucleic acid molecule havingthe nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), or a nucleic acidmolecule having a sequence complementary thereto. Such isolatedmolecules, particularly DNA molecules, are useful, for example, asprobes for gene mapping by in situ hybridization with chromosomes, andfor detecting expression of the TR21 gene in human tissue, for instance,by Northern blot analysis.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatedDNA molecule having the nucleotide sequence of the nucleotide sequenceshown in FIG. 1 (SEQ ID NO:1) is intended DNA fragments at least about15 nt, and more preferably at least about 20 nt, at least about 24 nt,still more preferably at least about 30 nt, at least about 35 nt, andeven more preferably, at least about 40 nt, at least about 45 nt, atleast about 50 nt, at least about 55 nt, at least about 60 nt, at leastabout 65 nt, at least about 70 nt, at least about 75 nt, at least about100 nt, at least about 150 nt, at least about 200 nt, at least about 250nt, at least about 300 nt in length which are useful, for example, asdiagnostic probes and primers as discussed herein. Of course, largerfragments 350-899 nt in length are also useful according to the presentinvention, as are fragments corresponding to most, if not all, of thenucleotide sequence as shown in FIG. 1 (SEQ ID NO:1), or thecomplementary strand thereto. By a fragment at least 20 nt in length,for example, is intended fragments which include 20 or more contiguousbases from the nucleotide sequence of the nucleotide sequence as shownin FIG. 1 (SEQ ID NO:1). In this context “about” includes theparticularly recited size, and sizes larger or smaller by several (5, 4,3, 2, or 1) nucleotides, at either terminus or at both termini. Inspecific embodiments, the fragments of the invention comprise, oralternatively consist of, nucleotides 12-50, 120-140, 156-164, 180-188,315-341, 348-371, 411-419, 444-464, and/or 537-551 of FIG. 1 (SEQ IDNO:1) or the complementary strand thereto. Polypeptides encoded by thesepolynucleotide are also encompassed.

Representative examples of TR21 polynucleotide fragments of theinvention include, for example, fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to 11, 12to 50, 51 to 59, 60 to 83, 84 to 110, 111 to 119, 120 to 140, 141 to155, 156 to 164, 165 to 179, 180 to 188, 189 to 215, 216 to 248, 249 to308, 309 to 314, 315 to 341, 342 to 347, 348 to 371, 372 to 411, 412 to419, 420 to 443, 444 to 464, 465 to 500, 501 to 536, 537 to 551, 552 to557, 558 to 590, 591 to 649, 650 to 708, 709 to 767, 768 to 826, 827 to885, and/or 886 to 899 of FIG. 1 (SEQ ID NO:1), or the complementarystrand thereto. In this context “about” includes the particularlyrecited ranges, and ranges larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini.

In specific embodiments, the polynucleotide fragments of the inventioncomprise, or alternatively, consist of, a sequence from nucleotide 60 to110, of FIG. 1 (SEQ ID NO:1), or the complementary strand thereto.

Preferably, the polynucleotide fragments of the invention encode apolypeptide which demonstrates a TR21 functional activity. By apolypeptide demonstrating a TR21 “functional activity” is meant, apolypeptide capable of displaying one or more known functionalactivities associated with a full-length (complete) TR21 protein. Suchfunctional activities include, but are not limited to, biologicalactivity, antigenicity (ability to bind (or compete with a TR21polypeptide for binding) to an anti-TR21 antibody), immunogenicity(ability to generate antibody which binds to a TR21 polypeptide),ability to form multimers with TR21 polypeptides of the invention, andability to bind to a receptor or ligand for a TR21 polypeptide (e.g.,Neutrokine-alpha (International Publication Number WO 98/18921)).

The functional activity of TR21 polypeptides, fragments, variants,derivatives, and analogs thereof, can be assayed by various methods.

For example, in one embodiment where one is assaying for the ability tobind or compete with full-length TR21 polypeptides for binding toanti-TR21 antibody, various immunoassays known in the art can be used,including but not limited to, competitive and non-competitive assaysystems using techniques such as radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitation reactions, immunodiffusion assays, in situimmunoassays (using colloidal gold, enzyme or radioisotope labels, forexample), western blots, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hemagglutination assays), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

In another embodiment, where a TR21 ligand is identified (e.g.,Neutrokine-alpha), or the ability of a polypeptide fragment, variant orderivative of the invention to multimerize is being evaluated, bindingcan be assayed by means well-known in the art, such as, for example,reducing and non-reducing gel chromatography, protein affinitychromatography, and affinity blotting. See generally, Phizicky, E., etal., Microbiol. Rev. 59:94-123 (1995). In another embodiment,physiological correlates of TR21 binding to its substrates (signaltransduction) can be assayed.

In addition, assays described herein (and otherwise known in the art mayroutinely be applied to measure the ability of TR21 polypeptides andfragments, variants derivatives and analogs thereof to elicit TR21related biological activity. For example, techniques described herein(see e.g., Examples 16, 17 and 18) and otherwise known in the art may beapplied or routinely modified to assay for the ability of thecompositions of the invention (e.g., fusion proteins comprising aportion of the extracellular domain of TR21 and an immunoglobulin Fcdomain) to inhibit or stimulate B cell proliferation (e.g.,Neutrokine-alpha mediated B cell proliferation).

Other methods will be known to the skilled artisan and are within thescope of the invention.

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding a member selected from the group: apolypeptide comprising or alternatively, consisting of, the TR21receptor extracellular domain (amino acid residues from about 1 to about81 in FIG. 1 (SEQ ID NO:2); a polypeptide comprising, or alternativelyconsisting of, the TR21 cysteine rich domain (amino acid residues fromabout 19 to about 35 in FIG. 1 (SEQ ID NO:2); a polypeptide comprising,or alternatively consisting of the TR21 transmembrane domain (amino acidresidues from about 82 to about 101 in FIG. 1 (SEQ ID NO:2); and/or apolypeptide comprising, or alternatively consisting of, the TR21intracellular domain (amino acid residues from about 102 to about 184 inFIG. 1 (SEQ ID NO:2)). Since the locations of these domains have beenpredicted by computer analysis, one of ordinary skill would appreciatethat the amino acid residues constituting these domains may varyslightly (e.g., by about 1 to 15 amino acid residues) depending on thecriteria used to define each domain.

Preferred nucleic acid fragments of the invention encode a full-lengthTR21 polypeptide lacking the nucleotides encoding the amino terminalmethionine in FIG. 1 (SEQ ID NO:1), as it is known that the methionineis cleaved naturally and such sequences may be useful in geneticallyengineering TR21 expression vectors. Polypeptides encoded by suchpolynucleotides are also contemplated by the invention.

Preferred nucleic acid fragments of the present invention furtherinclude nucleic acid molecules encoding epitope-bearing portions of theTR21 receptor proteins. In particular, such nucleic acid fragments ofthe present invention include nucleic acid molecules encoding: apolypeptide comprising amino acid residues from about 3 to about 15 inFIG. 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues fromabout 39 to about 45 in FIG. 1 (SEQ ID NO:2); a polypeptide comprisingamino acid residues from about 51 to about 53 in FIG. 1 (SEQ ID NO:2); apolypeptide comprising amino acid residues from about 59 to about 61 inFIG. 1 (SEQ ID NO:2); a polypeptide comprising amino acid residues fromabout 104 to about 112 in FIG. 1 (SEQ ID NO:2); a polypeptide comprisingamino acid residues from about 115 to about 122 in FIG. 1 (SEQ ID NO:2);a polypeptide comprising amino acid residues from about 136 to about 138in FIG. 1 (SEQ ID NO:2); a polypeptide comprising amino acid residuesfrom about 147 to about 153 in FIG. 1 (SEQ ID NO:2); and a polypeptidecomprising amino acid residues from about 178 to about 182 in FIG. 1(SEQ ID NO:2). In this context “about” includes the particularly recitedranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1)nucleotides, at either terminus or at both termini. The inventors havedetermined that the above polypeptide fragments are antigenic regions ofTR21. Methods for determining other such epitope-bearing portions of theTR21 proteins are described in detail below.

It is believed that the extracellular cysteine rich motif of TR21disclosed in FIG. 1 are important for interactions between TR21 and itsligands (e.g., Neutrokine alpha). Accordingly, specific embodiments ofthe invention are directed to polynucleotides encoding polypeptideswhich comprise, or alternatively consist of, the amino acid sequence ofamino acid residues 19 to 35 of FIG. 1 (SEQ ID NO:2). Polypeptidesencoded by these polynucleotides are also encompassed by the invention.

In additional embodiments, the polynucleotides of the invention encodefunctional attributes of TR21. Preferred embodiments of the invention inthis regard include fragments that comprise alpha-helix and alpha-helixforming regions (“alpha-regions”), beta-sheet and beta-sheet formingregions (“beta-regions”), turn and turn-forming regions(“turn-regions”), coil and coil-forming regions (“coil-regions”),hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions andantigenic regions of TR21.

The data representing the structural or functional attributes of TR21set forth in FIG. 2 and/or Table I, as described above, was generatedusing the various modules and algorithms of the DNA*STAR set on defaultparameters. In a preferred embodiment, the data presented in columnsVIII, XI, XIII and XIV of Table I can be used to determine regions ofTR21 which exhibit a high degree of potential for antigenicity. Regionsof high antigenicity are determined from the data presented in columnsVIII, XI, XIII and/or XIV by choosing values which represent regions ofthe polypeptide which are likely to be exposed on the surface of thepolypeptide in an environment in which antigen recognition may occur inthe process of initiation of an immune response.

Certain preferred regions in these regards are set out in FIG. 2, butmay, as shown in Table I, be represented or identified by using tabularrepresentations of the data presented in FIG. 2. The DNA*STAR computeralgorithm used to generate FIG. 2 (set on the original defaultparameters) was used to present the data in FIG. 2 in a tabular format(See Table I). The tabular format of the data in FIG. 2 may be used toeasily determine specific boundaries of a preferred region.

The above-mentioned preferred regions set out in FIG. 2 and in Table I,include, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequences set out in FIG. 1. Asset out in FIG. 2 and in Table I, such preferred regions includeGarnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions,Kyte-Doolittle hydrophilic regions, Hopp-Woods hydrophobic regions,Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexibleregions, Jameson-Wolf regions of high antigenic index and Eminisurface-forming regions.

TABLE I Res Pos I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 . .B . . . . 1.01 −0.67 * * . 0.95 1.41 Arg 2 . . B . . . . 1.51 −0.67 * *. 1.29 1.70 Arg 3 . . B . . . . 1.60 −1.10 * . . 1.63 2.61 Gly 4 . . . .. T C 1.18 −1.14 * * . 2.37 3.53 Pro 5 . . . . . T C 1.68 −1.07 * * F2.86 1.49 Arg 6 . . . . T T . 1.93 −1.07 * * F 3.40 1.49 Ser 7 . . B . .T . 1.93 −0.64 * * F 2.66 1.49 Leu 8 . . B . . . . 1.82 −1.07 * * F 2.461.88 Arg 9 . . B . . . . 1.58 −1.50 . * F 2.46 1.61 Gly 10 . . . . T T .1.58 −1.00 . * F 3.06 1.21 Arg 11 . . . . T T . 0.88 −0.96 . * F 3.062.27 Asp 12 . . . . T T . 0.97 −1.14 . * F 3.40 1.17 Ala 13 . . . . . TC 1.47 −0.71 . * F 2.86 1.83 Pro 14 . . . . . . C 1.14 −0.66 . * F 2.321.35 Ala 15 . . . . . T C 0.82 −0.23 * . F 1.88 1.25 Pro 16 . . . . . TC −0.14 0.34 * . F 0.79 0.66 Thr 17 . . B . . T . −0.36 0.49 . . F −0.050.32 Pro 18 . . . . T T . −0.36 0.49 . * F 0.35 0.49 Cys 19 . . B . . .. −0.14 0.49 . * . −0.40 0.32 Val 20 . A B . . . . −0.22 0.06 . . .−0.30 0.38 Pro 21 . A B . . . . −0.71 0.14 . . . −0.30 0.13 Ala 22 . A B. . . . −0.40 0.50 . . . −0.60 0.21 Glu 23 A A . . . . . −1.00 −0.07 * .. 0.30 0.48 Cys 24 A A . . . . . −1.14 −0.03 * . . 0.30 0.26 Phe 25 A A. . . . . −1.14 0.23 * * . −0.30 0.21 Asp 26 A A . . . . . −0.820.37 * * . −0.30 0.09 Leu 27 A A . . . . . −0.27 0.37 * * . −0.30 0.33Leu 28 A A . . . . . −0.93 0.30 * * . −0.30 0.52 Val 29 A A . . . . .−1.12 0.09 * * . −0.30 0.17 Arg 30 A A . . . . . −1.01 0.73 * * . −0.600.15 His 31 A A . . . . . −1.68 0.54 * * . −0.60 0.18 Cys 32 . A B . . .. −1.21 0.43 . * . −0.60 0.13 Val 33 . A B . . . . −1.21 0.21 . * .−0.30 0.07 Ala 34 . A B . . . . −1.17 0.90 . * . −0.60 0.04 Cys 35 . A B. . . . −1.17 1.09 . * . −0.60 0.06 Gly 36 . A B . . . . −1.44 0.51 * *. −0.60 0.16 Leu 37 . A B . . . . −0.99 0.36 * . . 0.00 0.23 Leu 38 . AB . . . . −0.02 0.29 * . . 0.30 0.67 Arg 39 . A B . . . . 0.36 −0.29 * *F 1.50 1.33 Thr 40 . . B . . T . 1.07 −0.29 * * F 2.20 2.50 Pro 41 . . .. . T C 1.20 −0.97 * . F 3.00 6.07 Arg 42 . . . . . T C 1.42 −1.23 * * F2.70 4.79 Pro 43 . . B . . T . 1.89 −0.73 * . F 2.20 3.35 Lys 44 . . . .. . C 1.19 −0.79 * . F 1.90 2.15 Pro 45 . . . . . . C 1.20 −0.71 . * F1.60 1.11 Ala 46 . . . . T . . 1.11 −0.33 . * F 1.05 0.96 Gly 47 . . B .. T . 0.79 −0.37 . * F 0.85 0.64 Ala 48 . . B . . T . 0.41 0.06 . . F0.49 0.64 Ser 49 . . . . . T C 0.16 0.13 * . F 0.93 0.64 Ser 50 . . . .. T C 0.48 0.06 * . F 1.32 1.00 Pro 51 . . . . . . C 0.76 −0.37 * . F1.96 1.95 Ala 52 . . . . . T C 0.51 −0.39 * * F 2.40 2.10 Pro 53 . . . .. T C 0.29 −0.27 * * F 2.16 1.58 Arg 54 . . B . . T . 0.59 0.03 * * F0.97 0.84 Thr 55 . . B . . T . 0.68 0.00 * * F 1.48 1.45 Ala 56 . . B .. . . 0.89 −0.07 * * F 1.24 1.45 Leu 57 . . B . . . . 1.48 −0.10 * * F1.20 1.28 Gln 58 . . B . . . . 1.39 −0.10 * * F 1.40 1.53 Pro 59 . . B .. T . 0.42 −0.20 * * F 1.80 2.03 Gln 60 . . B . . T . 0.39 −0.06 . * F2.00 1.83 Glu 61 . . B . . T . 0.39 −0.31 . * F 1.80 1.05 Ser 62 . . B .. T . 0.86 −0.21 . * F 1.45 0.68 Val 63 . . B . . . . 0.27 −0.21 . . F1.05 0.39 Gly 64 . . . . . . C 0.13 −0.11 . . . 0.90 0.23 Ala 65 . . . .. . C 0.13 0.31 . . . 0.10 0.17 Gly 66 . . . . . T C −0.46 −0.07 * . .0.90 0.39 Ala 67 A . . . . T . −0.74 −0.21 . . F 0.85 0.40 Gly 68 A . .. . T . −0.70 −0.14 . . F 0.85 0.40 Glu 69 A . . . . T . −0.57 0.04 . *. 0.10 0.33 Ala 70 . A B . . . . −0.79 0.04 . * . −0.30 0.51 Ala 71 . AB . . . . −0.66 0.23 . * . −0.30 0.43 Leu 72 . A B . . . . −0.41 0.23. * . −0.30 0.38 Pro 73 . A B . . . . −0.88 0.66 . * . −0.60 0.37 Leu 74. . B . . T . −1.69 0.84 . * . −0.20 0.30 Pro 75 . . B . . T . −1.801.03 . . F −0.05 0.30 Gly 76 . . B . . T . −1.56 1.13 . . . −0.20 0.17Leu 77 . . B . . T . −1.33 1.13 . . . −0.20 0.20 Leu 78 . . B B . . .−1.33 0.94 . . . −0.60 0.13 Phe 79 . . B B . . . −1.11 0.94 . . . −0.600.21 Gly 80 . . B B . . . −1.71 1.01 . . . −0.60 0.26 Ala 81 . A B . . .. −2.18 1.01 . . . −0.60 0.26 Pro 82 A A . . . . . −1.71 1.01 . . .−0.60 0.24 Ala 83 A A . . . . . −1.71 0.66 . . . −0.60 0.24 Leu 84 A A .. . . . −1.60 0.91 . * . −0.60 0.20 Leu 85 A A . . . . . −2.07 0.91 . .. −0.60 0.13 Gly 86 A A . . . . . −2.33 1.17 . . . −0.60 0.11 Leu 87 A A. . . . . −2.93 1.31 . . . −0.60 0.10 Ala 88 A A . . . . . −2.93 1.31 .. . −0.60 0.10 Leu 89 A A . . . . . −2.93 1.13 . . . −0.60 0.10 Val 90 AA . . . . . −2.98 1.39 . . . −0.60 0.10 Leu 91 A A . . . . . −3.44 1.34. . . −0.60 0.07 Ala 92 A A . . . . . −3.49 1.53 . . . −0.60 0.07 Leu 93. A B . . . . −3.24 1.49 . . . −0.60 0.07 Val 94 . A B . . . . −3.241.27 . . . −0.60 0.09 Leu 95 . A B . . . . −3.24 1.27 . . . −0.60 0.07Val 96 . A B . . . . −2.73 1.41 . . . −0.60 0.06 Gly 97 . A B . . . .−2.43 1.11 . * . −0.60 0.11 Leu 98 . . B B . . . −1.51 1.39 * . . −0.600.15 Val 99 . . B B . . . −0.54 0.70 * * . −0.60 0.38 Ser 100 . . B B .. . 0.38 0.06 . * . −0.30 0.76 Trp 101 . . B B . . . 1.23 −0.37 * * .0.45 1.80 Arg 102 . . B B . . . 1.69 −0.66 * * F 0.90 4.20 Arg 103 . . BB . . . 2.61 −1.30 * * F 0.90 6.14 Arg 104 . . . B T . . 2.66 −1.69 * *F 1.60 11.44 Gln 105 . . B B . . . 3.07 −1.91 * * F 1.50 4.82 Arg 106 .. B . . . . 3.01 −1.91 * * F 2.00 4.82 Arg 107 . . B . . . . 2.31−1.49 * * F 2.30 2.43 Leu 108 . . . . T . . 1.90 −0.99 * * F 3.00 1.42Arg 109 . . . . T . . 1.49 −1.00 . * F 2.55 0.97 Gly 110 . . . . . T C0.90 −0.61 * * F 2.25 0.66 Ala 111 . . . . . T C 0.79 −0.11 * * F 1.650.81 Ser 112 . . . . . T C 0.09 −0.80 . * F 1.65 0.72 Ser 113 . . . . .T C 0.69 −0.30 . * F 1.05 0.74 Ala 114 . . B . . . . 0.58 −0.30 . * F1.14 1.13 Glu 115 . . B . . . . 0.58 −0.80 . . F 1.78 1.40 Ala 116 A . .. . T . 1.17 −0.76 . . F 2.32 1.04 Pro 117 A . . . . T . 1.51 −1.14 . .F 2.66 1.71 Asp 118 . . . . T T . 1.81 −1.64 * . F 3.40 1.98 Gly 119 . .. . T T . 1.81 −1.64 * . F 3.06 3.27 Asp 120 . . . . T . . 1.60 −1.64 *. F 2.52 2.13 Lys 121 . . . . . . C 2.19 −1.64 * . F 1.98 1.98 Asp 122 .. . . . . C 2.19 −1.64 * . F 1.64 3.46 Ala 123 A . . . . T . 1.38−1.64 * * F 1.30 3.20 Pro 124 A . . . . T . 1.72 −0.96 * . F 1.30 1.32Glu 125 A . . . . T . 1.77 −0.96 * . F 1.30 1.32 Pro 126 A . . . . T .0.87 −0.96 * . F 1.30 2.61 Leu 127 A . . B . . . −0.02 −0.81 * . F 0.901.25 Asp 128 A . . B . . . −0.32 −0.56 * . F 0.75 0.51 Lys 129 A . . B .. . −0.92 0.13 * . F −0.15 0.23 Val 130 . . B B . . . −1.22 0.39 * . .−0.30 0.23 Ile 131 . . B B . . . −1.22 0.09 . . . −0.30 0.18 Ile 132 . .B B . . . −0.76 0.51 * . . −0.60 0.14 Leu 133 . . B B . . . −1.64 0.94 .. . −0.39 0.19 Ser 134 . . B . . T . −1.99 0.99 . . . 0.22 0.19 Pro 135. . . . . T C −1.13 0.69 * . F 0.78 0.36 Gly 136 . . . . T T . −0.830.00 . . F 2.09 0.74 Ile 137 . . . . . T C −0.26 −0.19 * . F 2.10 0.56Ser 138 . . . . . . C −0.03 −0.09 * . F 1.69 0.52 Asp 139 . A B . . . .0.06 −0.01 . . F 1.08 0.53 Ala 140 . A B . . . . −0.32 −0.01 . . F 1.021.17 Thr 141 . A B . . . . −0.27 −0.20 . . F 0.66 0.88 Ala 142 . A B . .. . 0.41 0.33 * . . −0.30 0.55 Pro 143 . . . . . . C 0.50 0.76 . . .−0.20 0.85 Ala 144 . . . . T . . 0.29 0.69 . . . 0.00 0.91 Trp 145 . . .. . . C 0.53 0.63 . . . 0.29 1.39 Pro 146 . . . . . . C 0.84 0.56 . . F0.63 0.89 Pro 147 . . . . . T C 1.43 0.13 . . F 1.62 1.53 Pro 148 . . .. T T . 1.43 −0.37 . . F 2.76 2.43 Gly 149 . . . . T T . 1.68 −0.86 . .F 3.40 2.43 Glu 150 . . . . . T C 1.66 −0.86 * . F 2.86 1.55 Asp 151 . .. . . T C 1.56 −0.80 . . F 2.52 1.45 Pro 152 . . . . T T . 1.56 −0.74 .. F 2.38 2.11 Gly 153 . . . . T T . 1.56 −0.74 . . F 2.20 1.89 Thr 154 .. B . . T . 1.56 −0.31 . . F 1.32 1.75 Thr 155 . . . . . . C 1.52 0.11 .. F 0.88 1.12 Pro 156 . . . . . T C 1.22 0.19 . . F 1.24 1.54 Pro 157 .. . . T T . 0.58 0.14 . . F 1.60 1.43 Gly 158 . . . . T T . 0.71 0.30 .. F 1.29 0.73 His 159 . . B . . T . 0.17 0.24 . . . 0.58 0.73 Ser 160 .. B B . . . 0.27 0.46 . . . −0.28 0.35 Val 161 . . B B . . . −0.11 0.46. * . −0.44 0.55 Pro 162 . . B B . . . −0.21 0.53 . . . −0.60 0.41 Val163 . . B B . . . 0.13 0.51 * . . −0.60 0.44 Pro 164 . . B . . . . −0.640.13 * . . 0.05 1.03 Ala 165 . . B . . . . −0.69 0.17 . . F 0.05 0.55Thr 166 A . . . . . . −0.13 0.17 . . F 0.05 0.73 Glu 167 A . . . . . .−0.23 −0.09 . . F 0.65 0.63 Leu 168 A . . . . . . 0.62 −0.03 . . F 0.650.90 Gly 169 A . . . . . . 0.02 −0.53 . . F 1.10 1.09 Ser 170 A . . . .. . −0.24 −0.33 . . F 0.65 0.52 Thr 171 A . . B . . . −0.24 0.31 . . F−0.15 0.47 Glu 172 A . . B . . . −0.56 0.11 . . F −0.15 0.68 Leu 173 A .. B . . . 0.30 0.17 . . F −0.15 0.73 Val 174 . . B B . . . 0.33 −0.21 .. F 0.60 1.01 Thr 175 . . B B . . . 0.04 −0.21 . . F 0.45 0.84 Thr 176 .. B B . . . 0.01 0.29 . . F 0.30 1.03 Lys 177 . . . B . . C −0.20 0.03 .. F 0.80 1.38 Thr 178 . . . . . . C 0.61 −0.19 . . F 1.90 1.48 Ala 179 .. . . . . C 1.47 −0.67 . * F 2.50 1.77 Gly 180 . . . . . T C 1.78 −0.76. . F 3.00 1.53 Pro 181 . . . . . T C 1.70 −0.36 . . F 2.40 1.84 Glu 182A . . . . T . 1.27 −0.41 . . F 1.90 2.33 Gln 183 A . . . . T . 1.19−0.49 . . . 1.45 3.01 Gln 184 . . B . . . . 1.39 −0.49 . . . 0.95 2.49

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, thecomplementary strand of nucleotides 60 to 110, 249 to 308, 12 to 50, 120to 140, 156 to 164, 180 to 188, 315 to 341, 348 to 371, 412 to 419, 444to 464 and/or 537 to 551 of SEQ ID NO:1. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C. Polypeptidesencoded by these nucleic acids are also encompassed by the invention.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 nt of the reference polynucleotide. These are useful, forexample, as diagnostic probes and primers as discussed above and in moredetail below. By a portion of a polynucleotide of “at least 20 nt inlength,” for example, is intended 20 or more contiguous nucleotides fromthe nucleotide sequence of the reference polynucleotide (e.g., thenucleotide sequence as shown in FIG. 1 (SEQ ID NO:1). In this context“about” includes the particularly recited size, and sizes larger orsmaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus orat both termini.

In specific embodiments, the polynucleotides of the invention are lessthan 110000 kb, 50000 kb, 10000 kb, 1000 kb, 500 kb, 400 kb, 350 kb, 300kb, 250 kb, 200 kb, 175 kb, 150 kb, 125 kb, 100 kb, 50 kb, 40 kb, 30 kb,25 kb, 20 kb, 15 kb, 10 kb, 7.5 kb, or 5 kb in length.

In further embodiments, polynucleotides of the invention comprise atleast 15, at least 30, at least 50, at least 100, or at least 250, atleast 500, or at least 800 contiguous nucleotides of TR21 codingsequence, but consist of less than or equal to 100 kb, 75 kb, 50 kb, 30kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic DNA that flanks the5′ or 3′ coding nucleotide set forth in FIG. 1 (SEQ ID NO:1). In furtherembodiments, polynucleotides of the invention comprise at least 15, atleast 30, at least 50, at least 100, or at least 250, at least 500, orat least 800 contiguous nucleotides of TR21 and/or coding sequence, butdo not comprise all or a portion of any TR21 intron. In anotherembodiment, the nucleic acid comprising TR21 coding sequence does notcontain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ tothe TR21 gene in the genome). In other embodiments, the polynucleotidesof the invention do not contain the coding sequence of more than 1000,500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flankinggene(s).

As indicated, nucleic acid molecules of the present invention whichencode a TR21 polypeptide may include, but are not limited to, thecoding sequence for the mature polypeptide, by itself; the codingsequence for the mature polypeptide and additional sequences, such asthose encoding a leader or secretory sequence, such as a pre-, or pro-or prepro-protein sequence; the coding sequence of the maturepolypeptide, with or without the aforementioned additional codingsequences, together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing—including splicing and polyadenylationsignals, for example—ribosome binding and stability of mRNA; additionalcoding sequence which codes for additional amino acids, such as thosewhich provide additional functionalities. Thus, for instance, thepolypeptide may be fused to a marker sequence, such as a peptide, whichfacilitates purification of the fused polypeptide. In certain preferredembodiments of this aspect of the invention, the marker sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(Qiagen, Inc.), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The “HA” tag is another peptideuseful for purification which corresponds to an epitope derived from theinfluenza hemagglutinin protein, which has been described by Wilson etal., Cell 37:767-778 (1984). As discussed below, other such fusionproteins include the TR21 receptor fused to Fc at the N- or C-terminus.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs, orderivatives of the TR21 receptor. Variants may occur naturally, such asa natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions which may involve one or more nucleotides. Thevariants may be altered in coding or non-coding regions or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions, or additions.Especially preferred among these are silent substitutions, additions,and deletions, which do not alter the properties and activities of theTR21 receptor or portions thereof. Also especially preferred in thisregard are conservative substitutions.

Further embodiments of the invention include isolated nucleic acidmolecules comprising, or alternatively consisting of, a polynucleotidehaving a nucleotide sequence at least 80%, 85%, or 90% identical, andmore preferably at least 95%, 96%, 97%, 98%, or 99% identical to: (a) anucleotide sequence encoding the polypeptide having the amino acidsequence shown in FIG. 1 (SEQ ID NO:2); (b) a nucleotide sequenceencoding the polypeptide having the amino acid sequence in FIG. 1 (SEQID NO: 2), but lacking the amino terminal methionine; (c) a nucleotidesequence encoding the polypeptide having the amino acid sequence atpositions 1 to 184 in FIG. 1 (SEQ ID NO:2); (d) a nucleotide sequenceencoding the TR21 extracellular domain; (e) a nucleotide sequenceencoding the TR21 cysteine-rich motif (i.e., amino acid residues about19 to about 35 in FIG. 1 (SEQ ID NO:2)); (f) a nucleotide sequenceencoding the TR21 transmembrane domain; (g) a nucleotide sequenceencoding the TR21 receptor intracellular domain; (h) a nucleotidesequence encoding the TR21 receptor extracellular and intracellulardomains with all or part of the transmembrane domain deleted; and (i) anucleotide sequence complementary to any of the nucleotide sequences in(a), (b), (c), (d), (e), (f), (g), or (h) above. Polypeptides encoded bythese polynucleotides are also encompassed by the invention.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a TR21polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five mismatches per each 100nucleotides of the reference nucleotide sequence encoding the TR21polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mismatches of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. The reference (query) sequence may be the entireTR21 encoding nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), or anyTR21 polynucleotide fragment (e.g., a polynucleotide encoding the aminoacid sequence of any of the TR21 N- and/or C-terminal deletionsdescribed herein), variant, derivative or analog, as described herein.

As a practical matter, whether any particular nucleic acid molecule isat least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) can bedetermined conventionally using known computer programs such as theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). Bestfit uses the local homology algorithmof Smith and Waterman, Advances in Applied Mathematics 2: 482-489(1981), to find the best segment of homology between two sequences. Whenusing Bestfit or any other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, such that the percentage of identity is calculatedover the full length of the reference nucleotide sequence and that gapsin homology of up to 5% of the total number of nucleotides in thereference sequence are allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions, a manual correction ismade to the results to take into consideration the fact that the FASTDBprogram does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.A determination of whether a nucleotide is matched/aligned is determinedby results of the FASTDB sequence alignment. This percentage is thensubtracted from the percent identity, calculated by the above FASTDBprogram using the specified parameters, to arrive at a final percentidentity score. This corrected score is what is used for the purposes ofthis embodiment. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score. For example, a 90 basesubject sequence is aligned to a 100 base query sequence to determinepercent identity. The deletions occur at the 5′ end of the subjectsequence and therefore, the FASTDB alignment does not show amatched/alignment of the first 10 bases at 5′ end. The 10 unpaired basesrepresent 10% of the sequence (number of bases at the 5′ and 3′ ends notmatched/total number of bases in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 bases were perfectly matched the finalpercent identity would be 90%. In another example, a 90 base subjectsequence is compared with a 100 base query sequence. This time thedeletions are internal deletions so that there are no bases on the 5′ or3′ of the subject sequence which are not matched/aligned with the query.In this case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only bases 5′ and 3′ of the subject sequencewhich are not matched/aligned with the query sequence are manuallycorrected for. No other manual corrections are made for the purposes ofthis embodiment.

The present application is directed to nucleic acid moleculescomprising, or alternatively consisting of a nucleotide sequence atleast 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to thenucleic acid sequence for example, shown in FIG. 1 (SEQ ID NO:1),irrespective of whether they encode a polypeptide having TR21 receptoractivity. This is because even where a particular nucleic acid moleculedoes not encode a polypeptide having TR21 functional activity, one ofskill in the art would still know how to use the nucleic acid molecule,for instance, as a hybridization probe or a polymerase chain reaction(PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having TR21 receptor activityinclude, inter alia: (1) isolating the TR21 gene or allelic variantsthereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) tometaphase chromosomal spreads to provide precise chromosomal location ofthe TR21 receptor gene, as described in Verma et al., Human Chromosomes:A Manual of Basic Techniques, Pergamon Press, New York (1988); and (3)Northern Blot analysis for detecting TR21 receptor mRNA expression inspecific tissues.

Preferred, however, are nucleic acid molecules comprising, oralternatively consisting of, a nucleotide sequence at least 80%, 85%,90%, 92%, 95%, 96%, 97%, 98% or 99% identical to for example, thenucleic acid sequence shown in FIG. 1 (SEQ ID NO:1), which do, in fact,encode a polypeptide having TR21 functional activity. By “a polypeptidehaving TR21 functional activity” is intended polypeptides exhibitingactivity similar, but not necessarily identical, to an activity of theTR21 receptor of the invention (either the full-length protein or,preferably, the mature protein), as measured in a particular biologicalassay.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 80%, 85%, 90%, 92%,95%, 96%, 97%, 98%, or 99% identical to, for example, the nucleic acidshown in FIG. 1 (SEQ ID NO:1), will encode a polypeptide “havingTR21-short functional activity.” Similarly, a large number of thenucleic acid molecules having a sequence at least 80%, 85%, 90%, 92%,95%, 96%, 97%, 98%, or 99% identical to, for example, a nucleic acidsequence shown in FIG. 1, will encode a polypeptide “having TR21functional activity.” In fact, since degenerate variants of thesenucleotide sequences all encode the same polypeptide, this will be clearto the skilled artisan even without performing a biological assay. Itwill be further recognized in the art that, for such nucleic acidmolecules that are not degenerate variants, a reasonable number willalso encode a polypeptide having TR21 functional activity. This isbecause the skilled artisan is fully aware of amino acid substitutionsthat are either less likely or not likely to significantly effectprotein function (e.g., replacing one aliphatic amino acid with a secondaliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in J. U. Bowie et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), wherein the authors indicate that proteinsare surprisingly tolerant of amino acid substitutions.

TR21 Polynucleotide Assays

This invention is also related to the use of TR21 polynucleotides todetect complementary polynucleotides such as, for example, as adiagnostic reagent. Detection of a normal and mutated form of TR21associated with a dysfunction will provide a diagnostic tool that canadd or define a diagnosis of a disease or susceptibility to a diseasewhich results from under-expression over-expression or alteredexpression of TR21 (or a soluble form thereof), such as, for example,tumors or autoimmune disease.

Individuals carrying mutations in the TR21 gene may be detected at theDNA level by a variety of techniques. Nucleic acids for diagnosis may beobtained from a biological sample from a patient (e.g., a patient'scells, such as from blood, urine, saliva, tissue biopsy and autopsymaterial). The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR prior to analysis. (Saiki et al.,Nature 324:163-166 (1986)). RNA or cDNA may also be used in the sameways. As an example, PCR primers complementary to the nucleic acidencoding TR21 can be used to identify and analyze TR21 expression andmutations. For example, deletions and insertions can be detected by achange in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled TR21 RNA or alternatively, radiolabeled TR21 antisenseDNA sequences. Perfectly matched sequences can routinely bedistinguished from mismatched duplexes by techniques known in the art,such as, for example, RNase A digestion or by differences in meltingtemperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high-resolution gel electrophoresisusing techniques known in the art. DNA fragments of different sequencesmay be distinguished on denaturing formamide gradient gels in which themobilities of different DNA fragments are retarded in the gel atdifferent positions according to their specific melting or partialmelting temperatures (see, e.g., Myers et al., Science 230:1242 (1985)).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods which include, but are not limited to, hybridization, RNaseprotection, chemical cleavage, direct DNA sequencing or the use ofrestriction enzymes, (e.g., restriction fragment length polymorphisms(“RFLP”) and Southern blotting of genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

The invention also encompasses isolated nucleic acids encoding theabove-described TR21 polypeptides and proteins. Such polynucleotidesequences can routinely be determined using techniques known in the art.For example, the amino acid sequence of the TR21 polypeptides of theinvention can be routinely determined using techniques known in the art,such as via the Edman degradation technique. (See, e.g., Creighton,1983, “Proteins: Structures and Molecular Principles”, W.H. Freeman &Co., N.Y., pp. 34-49). The amino acid sequence obtained may be used as aguide for the generation of oligonucleotide mixtures that can be used toscreen for polynucleotide sequences encoding TR21 polypeptides.Screening may be accomplished, for example, by standard hybridization orPCR techniques. For example, polynucleotides encoding TR21 polypeptidesof the invention may be isolated by techniques known in the art, suchas, for example, by performing PCR using two degenerate oligonucleotideprimer pools designed on the basis of amino acid sequence of the TR21polypeptide of interest. Techniques for the generation ofoligonucleotide mixtures and the screening are well known. (See, e.g.,Ausubel, supra., and PCR Protocols: A Guide to Methods and Applications,1990, Innis, M. et al., eds. Academic Press, Inc., New York). Thetemplate for the reaction may be cDNA obtained by reverse transcriptionof mRNA prepared from, for example, human or non-human cell lines ortissue, such as B cells, known or suspected to express a TR21polypeptide.

The PCR product may be subcloned and sequenced to ensure that theamplified sequences encode a TR21 polypeptide. The PCR fragment may thenbe used to isolate a full-length cDNA clone by a variety of methods. Forexample, the amplified fragment may be labeled and used to screen a cDNAlibrary, such as a bacteriophage cDNA library. Alternatively, thelabeled fragment may be used to isolate genomic clones via the screeningof a genomic library.

PCR technology may also be utilized to isolate full-length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown, or suspected, to express the TR21 gene, such as, for example, Bcells). A reverse transcription reaction may be performed on the RNAusing an oligonucleotide primer specific for the most 5′ end of theamplified fragment for the priming of first strand synthesis. Theresulting RNA/DNA hybrid may then be “tailed” with guanines using astandard terminal transferase reaction, the hybrid may be digested withRNAase H, and second strand synthesis may then be primed with a poly-Cprimer. Thus, cDNA sequences upstream of the amplified fragment mayeasily be isolated. For a review of cloning strategies which may beused, see e.g., Sambrook et al., 1989, infra.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a TR21 polypeptide. According to this strategy,polypeptides expressed by the cloned cDNA are screened using standardantibody screening techniques in conjunction with antibodies raisedagainst the TR21 polypeptides of the invention. (For screeningtechniques, see, for example, Harlow, E. and Lane, eds., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Press, Cold SpringHarbor.) Additionally, screening can be accomplished by screening withlabeled Neutrokine-alpha proteins or fusion proteins, such as, forexample, those described herein. Library clones detected via theirreaction with such labeled compounds can be purified and subjected tosequence analysis according to methods well known to those of skill inthe art.

TR21 Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors and/or nucleic acids of theinvention and the production of TR21 polypeptides or fragments thereofby recombinant techniques.

Host cells can be genetically engineered to incorporate nucleic acidmolecules and express polypeptides of the present invention. Thepolynucleotides may be introduced alone or with other polynucleotides.Such other polynucleotides may be introduced independently,co-introduced or introduced joined to the polynucleotides of theinvention.

In accordance with the present invention the vector may be, for example,a plasmid vector, a single or double-stranded phage vector, a single ordouble-stranded RNA or DNA viral vector. Such vectors may be introducedinto cells as polynucleotides, preferably DNA, by well-known techniquesfor introducing DNA and RNA into cells. Viral vectors may be replicationcompetent or replication defective. In the latter case viral propagationgenerally will occur only in complementing host cells.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will preferablyinclude a translation initiating at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture and tetracycline orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells, such asSaccharomyces or Pichia; insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanomacells; and plant cells. Appropriate culture mediums and conditions forthe above-described host cells are known in the art.

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. The availability of drugswhich inhibit the function of the enzymes encoded by these selectablemarkers allows for selection of cell lines in which the vector sequenceshave been amplified after integration into the host cell's DNA. Anadvantage of glutamine synthase based vectors are the availability ofcell lines (e.g., the murine myeloma cell line, NSO) which are glutaminesynthase negative. Glutamine synthase expression systems can alsofunction in glutamine synthase expressing cells (e.g. Chinese HamsterOvary (CHO) cells) by providing additional inhibitor to prevent thefunctioning of the endogenous gene. A glutamine synthase expressionsystem and components thereof are detailed in PCT publications:WO87/04462; WO86/05807; WO89/01036; W089/10404; and WO91/06657 which arehereby incorporated in their entireties by reference herein.Additionally, glutamine synthase expression vectors that may be usedaccording to the present invention are commercially available fromsuppliers including, for example, Lonza Biologics, Inc. (Portsmouth,N.H.). Expression and production of monoclonal antibodies using a GSexpression system in murine myeloma cells is described in Bebbington etal., Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol.Prog. 11:1 (1995) which are herein incorporated by reference.

Among vectors preferred for use in bacteria include pHE4-5 (ATCC™Accession No. 209311; and variations thereof), pQE70, pQE60 and pQE-9,available from Qiagen; pBS vectors, Phagescript vectors, PBLUESCRIPT™vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from STRATAGENE™; andptrc99a, pKK223-3, pKK233-3, pDR540, pRITS available from PHARMACIA™.Preferred expression vectors for use in yeast systems include, but arenot limited to, pYES2, pY01, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andpA0815 (all available from Invitrogen, Carlsbad, Calif.). Amongpreferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSGavailable from STRATAGENE™; and pSVK3, pBPV, pMSG and pSVL availablefrom PHARMACIA™. Other suitable vectors will be readily apparent to theskilled artisan.

In one embodiment, the yeast Pichia pastoris is used to express TR21protein in a eukaryotic system. Pichia pastoris is a methylotrophicyeast which can metabolize methanol as its sole carbon source. A mainstep in the metabolism of methanol is the oxidation of methanol toformaldehyde using O₂. This reaction is catalyzed by the enzyme alcoholoxidase. In order to metabolize methanol as its sole carbon source,Pichia pastoris must generate high levels of alcohol oxidase due, inpart, to the relatively low affinity of alcohol oxidase for O₂.Consequently, in a growth medium depending on methanol as a main carbonsource, the promoter region of one of the two alcohol oxidase genes(AOX1) is highly active. In the presence of methanol, alcohol oxidaseproduced from the AOX1 gene comprises up to approximately 30% of thetotal soluble protein in Pichia pastoris. See, Ellis, S. B., et al.,Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77(1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987).Thus, a heterologous coding sequence, such as, for example, a TR21polynucleotide of the present invention, under the transcriptionalregulation of all or part of the AOXI regulatory sequence is expressedat exceptionally high levels in Pichia yeast grown in the presence ofmethanol.

In one example, the plasmid vector pPIC9K is used to express DNAencoding a TR21 polypeptide of the invention, as set forth herein, in aPichia yeast system essentially as described in “Pichia Protocols:Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. TheHumana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a TR21 protein of the invention by virtue ofthe strong AOX1 promoter linked to the Pichia pastoris alkalinephosphatase (PHO) secretory signal peptide (i.e., leader) locatedupstream of a multiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as,pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG as required.

In one embodiment, high-level expression of a heterologous codingsequence, such as, for example, a TR21 polynucleotide of the presentinvention, may be achieved by cloning the heterologous polynucleotide ofthe invention into an expression vector such as, for example, pGAPZ orpGAPZalpha, and growing the yeast culture in the absence of methanol.

The present invention also relates to host cells containing theabove-described vector constructs described herein, and additionallyencompasses host cells containing nucleotide sequences of the inventionthat are operably associated with one or more heterologous controlregions (e.g., promoter and/or enhancer) using techniques known of inthe art. The host cell can be a higher eukaryotic cell, such as amammalian cell (e.g., a human derived cell), or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. The host strain may modulate the expression of theinserted gene sequences, or modify and process the gene product in thespecific fashion desired. Expression from certain promoters can beelevated in the presence of certain inducers; thus expression of thegenetically engineered polypeptide may be controlled. Furthermore,different host cells have characteristics and specific mechanisms forthe translational and post-translational processing and modification(e.g., phosphorylation, cleavage) of proteins. Appropriate cell linescan be chosen to ensure the desired modifications and processing of theforeign protein expressed.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., TR21 coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with TR21 polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous TR21 polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous TR21 polynucleotide sequences via homologous recombination(see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; InternationalPublication Number WO 96/29411; International Publication Number WO94/12650; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989);and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of eachof which are incorporated by reference in their entireties).

The TR21 polypeptide may be expressed in a modified form, such as afusion protein (comprising the polypeptide joined via a peptide bond toa heterologous protein sequence (of a different protein)), and mayinclude not only secretion signals but also additional heterologousfunctional regions. Alternatively, such a fusion protein can be made byprotein synthetic techniques, e.g., by use of a peptide synthesizer.Thus, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence in the host cell, during purification orduring subsequent handling and storage. Also, peptide moieties may beadded to the polypeptide to facilitate purification. Such regions may beremoved prior to final preparation of the polypeptide. The addition ofpeptide moieties to polypeptides to engender secretion or excretion, toimprove stability and to facilitate purification, among others, arefamiliar and routine techniques in the art.

In one embodiment, polynucleotides encoding TR21 polypeptides of theinvention may be fused to signal sequences which will direct thelocalization of a protein of the invention to particular compartments ofa prokaryotic or eukaryotic cell and/or direct the secretion of aprotein of the invention from a prokaryotic or eukaryotic cell. Forexample, in E. coli, one may wish to direct the expression of theprotein to the periplasmic space. Examples of signal sequences orproteins (or fragments thereof) to which the polypeptides of theinvention may be fused in order to direct the expression of thepolypeptide to the periplasmic space of bacteria include, but are notlimited to, the pe1B signal sequence, the maltose binding protein (MBP)signal sequence, MBP, the ompA signal sequence, the signal sequence ofthe periplasmic E. coli heat-labile enterotoxin B-subunit, and thesignal sequence of alkaline phosphatase. Several vectors arecommercially available for the construction of fusion proteins whichwill direct the localization of a protein, such as the pMAL series ofvectors (particularly the pMAL-p series) available from New EnglandBiolabs. In a specific embodiment, polynucleotides encoding TR21polypeptides of the invention may be fused to the pe1B pectate lyasesignal sequence to increase the efficiency of expression andpurification of such polypeptides in Gram-negative bacteria. See, U.S.Pat. Nos. 5,576,195 and 5,846,818, the contents of which are hereinincorporated by reference in their entireties.

Examples of signal peptides that may be fused to a polypeptide of theinvention in order to direct its secretion in mammalian cells include,but are not limited to, the MPIF-1 signal sequence (amino acids 1-21 ofGenBank Accession number AAB51134), the stanniocalcin signal sequence(MLQNSAVLLLLVISASA, SEQ ID NO:4), and a consensus signal sequence(MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:5). A suitable signal sequence thatmay be used in conjunction with baculoviral expression systems is thegp67 signal sequence, (amino acids 1-19 of GenBank Accession NumberAAA72759).

A preferred fusion protein comprises a heterologous region fromimmunoglobulin that is useful to solubilize proteins. For example,EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteinscomprising various portions of constant region of immunoglobulinmolecules together with another human protein or part thereof. In manycases, the Fc part in a fusion protein is thoroughly advantageous foruse in therapy and diagnosis and thus results, for example, in improvedpharmacokinetic properties (EP-A 0232 262). On the other hand, for someuses, it would be desirable to be able to delete the Fc part after thefusion protein has been expressed, detected and purified in theadvantageous manner described. This is the case when the Fc portionproves to be a hindrance to use in therapy and diagnosis, for example,when the fusion protein is to be used as an antigen for immunizations.In drug discovery, for example, human proteins, such as thehIL5-receptor, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., Journal of Molecular Recognition 8:52-58 (1995) andK. Johanson et al., The Journal of Biological Chemistry 270:16:9459-9471(1995).

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or non-glycosylated. In addition, polypeptides of theinvention may also include an initial modified methionine residue, insome cases as a result of host-mediated processes.

In addition, proteins of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., N.Y. (1983),and Hunkapiller, et al., Nature 310:105-111 (1984)). For example, apolypeptide corresponding to a fragment of the TR21 polypeptides of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the TR21polypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

The invention additionally, encompasses TR21 polypeptides which aredifferentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited to, specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation,formylation, oxidation, reduction, metabolic synthesis in the presenceof tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor 0-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

In specific embodiments, TR21 polypeptides of the invention are attachedto macrocyclic chelators useful for conjugating radiometal ions,including but not limited to, ¹¹¹In, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, topolypeptides. In a preferred embodiment, the radiometal ion associatedwith the macrocyclic chelators attached to TR21 polypeptides of theinvention is ¹¹¹In. In another preferred embodiment, the radiometal ionassociated with the macrocyclic chelator attached to TR21 polypeptidesof the invention is ⁹⁰Y. In specific embodiments, the macrocyclicchelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″″-tetraacetic acid(DOTA). In other specific embodiments, the DOTA is attached to TR21polypeptide of the invention via a linker molecule. Examples of linkermolecules useful for conjugating DOTA to a polypeptide are commonlyknown in the art—see, for example, DeNardo et al., Clin Cancer Res.4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7,1999; and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50, 1999 which arehereby incorporated by reference in their entirety. In addition, U.S.Pat. Nos. 5,652,361 and 5,756,065, which disclose chelating agents thatmay be conjugated to antibodies, and methods for making and using them,are hereby incorporated by reference in their entireties. Though U.S.Pat. Nos. 5,652,361 and 5,756,065 focus on conjugating chelating agentsto antibodies, one skilled in the art could readily adapt the methoddisclosed therein in order to conjugate chelating agents to otherpolypeptides.

Also provided by the invention are chemically modified derivatives ofTR21 which may provide additional advantages such as increasedsolubility, stability and circulating time of the polypeptide, ordecreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemicalmoieties for derivatization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The polypeptides may be modified at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to a protein via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) of the protein or to morethan one type of amino acid residue (e.g., lysine, histidine, asparticacid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may beaccomplished by any number of means. For example, polyethylene glycolmay be attached to the protein either directly or by an interveninglinker. Linkerless systems for attaching polyethylene glycol to proteinsare described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998);U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO98/32466, the disclosures of each of which are incorporated herein byreference.

One system for attaching polyethylene glycol directly to amino acidresidues of proteins without an intervening linker employs tresylatedMPEG, which is produced by the modification of monomethoxy polyethyleneglycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Upon reaction ofprotein with tresylated MPEG, polyethylene glycol is directly attachedto amine groups of the protein. Thus, the invention includesprotein-polyethylene glycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number ofdifferent intervening linkers. For example, U.S. Pat. No. 5,612,460, theentire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol toproteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin WO 98/32466, the entire disclosure of which is incorporated herein byreference. Pegylated protein products produced using the reactionchemistries set out herein are included within the scope of theinvention.

The number of polyethylene glycol moieties attached to each protein ofthe invention (i.e., the degree of substitution) may also vary. Forexample, the pegylated proteins of the invention may be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

As mentioned, the TR21 proteins of the invention may be modified byeither natural processes, such as posttranslational processing, or bychemical modification techniques which are well known in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degrees at several sites in a given TR21polypeptide. TR21 polypeptides may be branched, for example, as a resultof ubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic TR21 polypeptides may result fromnatural posttranslational processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992)).

The TR21 polypeptides of the invention can be recovered and purifiedfrom chemical synthesis and recombinant cell cultures by standardmethods which include, but are not limited to, ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxyapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography (“HPLC”) is employed for purification. Well-knowntechniques for refolding protein may be employed to regenerate activeconformation when the polypeptide is denatured during isolation and/orpurification.

TR21 receptor polynucleotides and polypeptides may be used in accordancewith the present invention for a variety of applications, particularlythose that make use of the chemical and biological properties of TR21.Among these are applications in treatment of autoimmune diseases. Alsocontemplated are applications in the treatment of tumors, resistance toparasites, bacteria and viruses, to inhibit proliferation of B cells, toinduce proliferation of T-cells, endothelial cells and certainhematopoietic cells, to treat restenosis, graft vs. host disease, toregulate anti-viral responses and to prevent certain autoimmune diseasesafter stimulation of TR21 by an agonist. Additional applications relateto diagnosis and to treatment of disorders of cells, tissues andorganisms. These aspects of the invention are discussed further below.

TR21 Transgenics and “Knock-Outs”

The TR21 proteins of the invention can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows and non-human primates, e.g., baboons, monkeys, and chimpanzees maybe used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art, are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., nucleic acids of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, Mol Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pluripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety. Further, the contents of each of the documentsrecited in this paragraph is herein incorporated by reference in itsentirety. Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229(1989), which is incorporated by reference herein in its entirety. Seealso, U.S. Pat. No. 5,464,764 (Capecchi, et al., Positive-NegativeSelection Methods and Vectors); U.S. Pat. No. 5,631,153 (Capecchi, etal., Cells and Non-Human Organisms Containing Predetermined GenomicModifications and Positive-Negative Selection Methods and Vectors forMaking Same); U.S. Pat. No. 4,736,866 (Leder, et al., TransgenicNon-Human Animals); and U.S. Pat. No. 4,873,191 (Wagner, et al., GeneticTransformation of Zygotes); each of which is hereby incorporated byreference in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campbell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)), each ofwhich is herein incorporated by reference in its entirety).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric animals. The transgene may be integrated as a single transgeneor as multiple copies such as in concatamers, e.g., head-to-head tandemsor head-to-tail tandems. The transgene may also be selectivelyintroduced into and activated in a particular cell type by following,for example, the teaching of Lasko et al. (Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Science 265:103-106 (1994)). The regulatorysequences required for such a cell-type specific inactivation willdepend upon the particular cell type of interest, and will be apparentto those of skill in the art. The contents of each of the documentsrecited in this paragraph is herein incorporated by reference in itsentirety.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic and “knock-out” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of TR21 polypeptides, studyingconditions and/or disorders associated with aberrant TR21 expression,and in screening for compounds effective in ameliorating such conditionsand/or disorders.

In further embodiments of the invention, cells that are geneticallyengineered to express the proteins of the invention, or alternatively,that are genetically engineered not to express the proteins of theinvention (e.g., knockouts) are administered to a patient in vivo. Suchcells may be obtained from the patient (i.e., animal, including human)or an MHC compatible donor and can include, but are not limited tofibroblasts, bone marrow cells, blood cells (e.g., lymphocytes),adipocytes, muscle cells, endothelial cells, etc. The cells aregenetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally. Alternatively, the cells can be incorporated into amatrix and implanted in the body, e.g., genetically engineeredfibroblasts can be implanted as part of a skin graft; geneticallyengineered endothelial cells can be implanted as part of a lymphatic orvascular graft. (See, for example, Anderson et al. U.S. Pat. No.5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959, each of whichis incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well-known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

TR21 Receptor Polypeptides and Fragments

The TR21 proteins (polypeptides) of the invention may be in monomers ormultimers (i.e., dimers, trimers, tetramers, and higher multimers).Accordingly, the present invention relates to monomers and multimers ofthe TR21 proteins (polypeptides) of the invention, their preparation,and compositions (preferably, pharmaceutical compositions) containingthem. In specific embodiments, the polypeptides of the invention aremonomers, dimers, trimers or tetramers. In additional embodiments, themultimers of the invention are at least dimers, at least trimers, or atleast tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing only TR21proteins of the invention (including TR21 fragments, variants, andfusion proteins, as described herein). These homomers may contain TR21proteins having identical or different polypeptide sequences. In aspecific embodiment, a homomer of the invention is a multimer containingonly TR21 proteins having an identical polypeptide sequence. In anotherspecific embodiment, a homomer of the invention is a multimer containingTR21 proteins having different polypeptide sequences (e.g., TR21mutations containing proteins have polypetide sequences. In specificembodiments, the multimer of the invention is a homodimer (e.g.,containing TR21 proteins having identical or different polypeptidesequences) or a homotrimer (e.g., containing TR21 proteins havingidentical or different polypeptide sequences). In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containingheterologous proteins (i.e., proteins containing only polypeptidesequences that do not correspond to polypeptide sequences encoded by theTR21 gene) in addition to the TR21 proteins of the invention. In aspecific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when proteins of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when proteins of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the TR21 proteins of the invention.Such covalent associations may involve one or more amino acid residuescontained in the polypeptide sequence of the protein (e.g., thepolypeptide sequence shown in FIG. 1 (SEQ ID NO:2)). In one instance,the covalent associations are cross-linking between cysteine residueslocated within the polypeptide sequences of the proteins which interactin the native (i.e., naturally occurring) polypeptide. In anotherinstance, the covalent associations are the consequence of chemical orrecombinant manipulation. Alternatively, such covalent associations mayinvolve one or more amino acid residues contained in the heterologouspolypeptide sequence in a TR21 fusion protein. In one example, covalentassociations are mediated by the heterologous sequence contained in afusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925). Ina specific example, the covalent associations are mediated by theheterologous sequence contained in a TR21-Fc fusion protein of theinvention (as described herein). In another specific example, covalentassociations of fusion proteins of the invention are mediated byheterologous polypeptide sequences from other TNF family ligand/receptormembers that are capable of forming covalently associated multimers,such as for example, osteoprotegerin (see, e.g., InternationalPublication No. WO 98/49305, the contents of which are hereinincorporated by reference in its entirety). In another embodiment, twoor more TR21 polypeptides of the invention are joined through syntheticlinkers (e.g., peptide, carbohydrate or soluble polymer linkers).Examples include those peptide linkers described in U.S. Pat. No.5,073,627 (hereby incorporated by reference). Proteins comprisingmultiple TR21 polypeptides separated by peptide linkers may be producedusing conventional recombinant DNA technology.

Another method for preparing multimeric TR21 polypeptides of theinvention involves use of TR21 polypeptides fused to a leucine zipper orisoleucine zipper polypeptide sequence. Leucine zipper domains andisoleucine zipper domains are polypeptides that promote multimerizationof the proteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, (1988)), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimeric TR21proteins are those described in PCT application WO 94/10308, herebyincorporated by reference. Recombinant fusion proteins comprising asoluble TR21 polypeptide fused to a peptide that dimerizes or trimerizesin solution are expressed in suitable host cells, and the resultingsoluble multimeric TR21 is recovered from the culture supernatant usingtechniques known in the art.

Certain members of the TNF family of proteins are believed to exist intrimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al.,Cell 73:431, 1993). Thus, trimeric TR21 may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties arethose that preferentially form trimers. One example is a leucine zipperderived from lung surfactant protein D (SPD), as described in Hoppe etal. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser.No. 08/446,922, hereby incorporated by reference. Other peptides derivedfrom naturally occurring trimeric proteins may be employed in preparingtrimeric TR21.

In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequences contained in Flag®-TR21fusion proteins of the invention. In a further embodiment, associatedproteins of the invention are associated by interactions betweenheterologous polypeptide sequence contained in Flag®-TR21 fusionproteins of the invention and anti-Flag® antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, proteins desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the polypeptidesequence of the proteins desired to be contained in the multimer (see,e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by referencein its entirety). Further, proteins of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide sequence of the protein and techniquesknown in the art may be applied to generate multimers containing one ormore of these modified proteins (see, e.g., U.S. Pat. No. 5,478,925,which is herein incorporated by reference in its entirety).Additionally, techniques known in the art may be applied to generateliposomes containing the protein components desired to be contained inthe multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, proteinscontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain and which can beincorporated by membrane reconstitution techniques into liposomes (see,e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by referencein its entirety).

The polypeptides of the present invention are preferably provided in anisolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontained within a recombinant host cell is considered isolated forpurposes of the present invention. Also intended as an “isolatedpolypeptide” are polypeptides that have been purified, partially orsubstantially, from a recombinant host cell. For example, arecombinantly produced version of the TR21 polypeptide can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988).

Accordingly, in one embodiment, the invention provides an isolated TR21polypeptide having the amino acid sequence encoded by the amino acidsequence in FIG. 1 (SEQ ID NO:2), or a polypeptide comprising, oralternatively consisting of, a portion of the above polypeptides, suchas for example, a mature TR21, the TR21 extracellular domain (aminoacids 1 to 81 of FIG. 1 (SEQ ID NO:2)), the TR21 cysteine rich motif(amino acids 19 to 35 of FIG. 1 (SEQ ID NO:2)), and/or the TR21intracellular domain (amino acids 102 to 184 of FIG. 1 (SEQ ID NO:2)).

Polypeptide fragments of the present invention include polypeptidescomprising or alternatively, consisting of: an amino acid sequencecontained in FIG. 1 (SEQ ID NO:2); and encoded by a nucleic acid whichhybridizes (e.g., under stringent hybridization conditions) to thecomplementary strand of the nucleotide sequence shown in FIG. 1 (SEQ IDNO:1), or a fragment thereof. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Protein fragments may be “free-standing,” or comprised within a largerpolypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentsthat comprise or alternatively, consist of about amino acid residues: 1to 2, 3 to 15, 16 to 18, 19 to 26, 27 to 35, 36 to 38, 39 to 45, 46 to50, 51 to 53, 54 to 58, 59 to 61, 62 to 70, 71 to 81, 82 to 101, 102 to103, 104 to 112, 113 to 114, 115 to 122, 123 to 135, 136 to 138, 139 to146, 147 to 153, 154 to 177, 178 to 182 and/or 183 to 184, of SEQ IDNO:2 or FIG. 1. In this context “about” includes the particularlyrecited ranges, ranges larger or smaller by several (5, 4, 3, 2, or 1)amino acids, at either extreme or at both extremes. Moreover,polypeptide fragments can be at least 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150 or amino acids in length.Polynucleotides encoding these polypeptides are also encompassed by theinvention. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

In additional embodiments, the polypeptide fragments of the inventioncomprise, or alternatively consist of, one or more TR21 domains.Preferred polypeptide fragments of the present invention include one,two, three or more members selected from the group: (a) a polypeptidecomprising or alternatively, consisting of, the TR21 extracellulardomain (predicted to constitute amino acid residues 1 to 81 FIG. 1 (SEQID NO:2)); (b) a polypeptide comprising or alternatively, consisting of,the TR21 cysteine rich domain (predicted to constitute amino acidresidues 19 to 35 FIG. 1 (SEQ ID NO:2)); (c) a polypeptide comprising oralternatively, consisting of, the TR21 transmembrane domain (predictedto constitute amino acid residues 82 to 101 FIG. 1 (SEQ ID NO:2)); (d) apolypeptide comprising or alternatively, consisting of, the TR21intracellular domain (predicted to constitute amino acid residues 102 to184 FIG. 1 (SEQ ID NO:2)); (e) a polypeptide comprising, oralternatively, consisting of, one, two, three, four or more, epitopebearing portions of the TR21 protein; or (f) any combination ofpolypeptides (a)-(e). Polynucleotides encoding these polypeptides arealso encompassed by the invention.

As discussed above, it is believed that the extracellular cysteine richmotif of TR21 is important for interactions between TR21 and its ligands(e.g., Neutrokine-alpha). Accordingly, in preferred embodiments,polypeptides of the invention comprise, or alternatively consist ofamino acid residues 19 to 35 of FIG. 1 (SEQ ID NO:2). Proteinscomprising or alternatively consisting of a polypeptide sequence whichis at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical tothe polypeptide sequences of the cysteine rich motif are alsoencompassed by the invention. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Among the especially preferred fragments of the invention are fragmentscharacterized by structural or functional attributes of TR21. Suchfragments include amino acid residues that comprise alpha-helix andalpha-helix forming regions (“alpha-regions”), beta-sheet andbeta-sheet-forming regions (“beta-regions”), turn and turn-formingregions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., containing three or morecontiguous amino acids having an antigenic index of greater than orequal to 1.5, as identified using the default parameters of theJameson-Wolf program) of complete (i.e., full-length) TR21 (FIG. 1 (SEQID NO:2)). Certain preferred regions are those set out in FIG. 2 andTable 1 and include, but are not limited to, regions of theaforementioned types identified by analysis of the amino acid sequencedepicted in FIG. 1 (SEQ ID NO:2), such preferred regions include;Garnier-Robson predicted alpha-regions, beta-regions, turn-regions, andcoil-regions; Chou-Fasman predicted alpha-regions, beta-regions, andturn-regions; Kyte-Doolittle predicted hydrophilic; Hopp-Woods predictedhydrophobic regions; Eisenberg alpha and beta amphipathic regions; Eminisurface-forming regions; and Jameson-Wolf high antigenic index regions,as predicted using the default parameters of these computer programs.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

As mentioned above, even if deletion of one or more amino acids from theN-terminus of a protein results in modification of loss of one or morebiological functions of the protein, other functional activities (e.g.,biological activities, ability to multimerize, ability to bind TR21ligand (e.g., Neutrokine-alpha)) may still be retained. For instance,Ron et al., J. Biol. Chem., 268:2984-2988 (1993) reported modified KGFproteins that had heparin binding activity even if 3, 8, or 27amino-terminal amino acid residues were missing. The ability ofshortened TR21 “muteins” to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptides generallywill be retained when less than the majority of the residues of thecomplete or mature polypeptide are removed from the N-terminus. As usedherein, a “mutein” is a mutant protein including single or multipleamino acid substitutions, deletions, or additions (including fusionproteins). Whether a particular polypeptide lacking N-terminal residuesof a complete full-length polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that a TR21 muteinwith a large number of deleted N-terminal amino acid residues may retainsome biological or immunogenic activities. In fact, peptides composed ofas few as six TR21 amino acid residues may often evoke an immuneresponse.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the TR21 aminoacid sequence shown in FIG. 1, up to the alanine residue at positionnumber 179 and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising, oralternatively consisting of, the amino acid sequence of residues n¹-184of FIG. 1, where n¹ is an integer from 2 to 179 corresponding to theposition of the amino acid residue in FIG. 1 (SEQ ID NO:2).

More in particular, N-terminal deletions of the TR21 polypeptide of theinvention shown as SEQ ID NO:2 include polypeptides comprising the aminoacid sequence of residues: R-2 to Q-184; R-3 to Q-184; G-4 to Q-184; P-5to Q-184; R-6 to Q-184; S-7 to Q-184; L-8 to Q-184; R-9 to Q-184; G-10to Q-184; R-11 to Q-184; D-12 to Q-184; A-13 to Q-184; P-14 to Q-184;A-15 to Q-184; P-16 toQ-184; T-17 to Q-184; P-18 to Q-184; C-19 toQ-184; V-20 to Q-184; P-21 to Q-184; A-22 to Q-184; E-23 to Q-184; C-24to Q-184; F-25 to Q-184; D-26 to Q-184; L-27 to Q-184; L-28 to Q-184;V-29 to Q-184; R-30 to Q-184; H-31 to Q-184; C-32 to Q-184; V-33 toQ-184; A-34 to Q-184; C-35 to Q-184; G-36 to Q-184; L-37 to Q-184; L-38to Q-184; R-39 to Q-184; T-40 to Q-184; P-41 to Q-184; R-42 to Q-184;P-43 to Q-184; K-44 to Q-184; P-45 to Q-184; A-46 to Q-184; G-47 toQ-184; A-48 to Q-184; S-49 to Q-184; S-50 to Q-184; P-51 to Q-184; A-52to Q-184; P-53 to Q-184; R-54 to Q-184; T-55 to Q-184; A-56 to Q-184;L-57 to Q-184; Q-58 to Q-184; P-59 to Q-184; Q-60 to Q-184; E-61 toQ-184; S-62 to Q-184; V-63 to Q-184; G-64 to Q-184; A-65 to Q-184; G-66to Q-184; A-67 to Q-184; G-68 to Q-184; E-69 to Q-184; A-70 to Q-184;A-71 to Q-184; L-72 to Q-184; P-73 to Q-184; L-74 to Q-184; P-75 toQ-184; G-76 to Q-184; L-77 to Q-184; L-78 to Q-184; F-79 to Q-184; G-80to Q-184; A-81 to Q-184; P-82 to Q-184; A-83 to Q-184; L-84 to Q-184;L-85 to Q-184; G-86 to Q-184; L-87 to Q-184; A-88 to Q-184; L-89 toQ-184; V-90 to Q-184; L-91 to Q-184; A-92 to Q-184; L-93 to Q-184; V-94to Q-184; L-95 to Q-184; V-96 to Q-184; G-97 to Q-184; L-98 to Q-184;V-99 to Q-184; S-100 to Q-184; W-101 to Q-184; R-102 to Q-184; R-103 toQ-184; R-104 to Q-184; Q-105 to Q-184; R-106 to Q-184; R-107 to Q-184;L-108 to Q-184; R-109 to Q-184; G-110 to Q-184; A-111 to Q-184; S-112 toQ-184; S-113 to Q-184; A-114 to Q-184; E-115 to Q-184; A-116 to Q-184;P-117 to Q-184; D-118 to Q-184; G-119 to Q-184; D-120 to Q-184; K-121 toQ-184; D-122 to Q-184; A-123 to Q-184; P-124 to Q-184; E-125 to Q-184;P-126 to Q-184; L-127 to Q-184; D-128 to Q-184; K-129 to Q-184; V-130 toQ-184; 1-131 to Q-184; I-132 to Q-184; L-33 to Q-184; S-134 to Q-184;P-135 to Q-184; G-136 to Q-184; I-137 to Q-184; S-138 to Q-184; D-139 toQ-184; A-140 to Q-184; T-141 to Q-184; A-142 to Q-184; P-143 to Q-184;A-144 to Q-184; W-145 to Q-184; P-146 to Q-184; P-147 to Q-184; P-148 toQ-184; G-149 to Q-184; E-150 to Q-184; D-151 to Q-184; P-152 to Q-184;G-153 to Q-184; T-154 to Q-184; T-155 to Q-184; P-156 to Q-184; P-157 toQ-184; G-158 to Q-184; H-159 to Q-184; S-160 to Q-184; V-161 to Q-184;P-162 to Q-184; V-163 to Q-184; P-164 to Q-184; A-165 to Q-184; T-166 toQ-184; E-167 to Q-184; L-168 to Q-184; G-169 to Q-184; S-170 to Q-184;T-171 to Q-184; E-172 to Q-184; L-173 to Q-184; V-174 to Q-184; T-175 toQ-184; T-176 to Q-184; K-177 to Q-184; T-178 to Q-184; and/or A-179 toQ-184 of the TR21 sequence disclosed as SEQ ID NO:2 and shown in FIG. 1.Polypeptides encoded by these polynucleotides are also encompassed bythe invention.

In another embodiment, N-terminal deletions of the TR21 polypeptide canbe described by the general formula n²−81, where n² is a number from 2to 76, corresponding to the position of amino acid identified in FIG. 1.(SEQ ID NO:2). Preferably, N-terminal deletions of the TR21 polypeptideof the invention shown as FIG. 1 (SEQ ID NO:2) include polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of residues R-2 to A-81; R-3 to A-81; G-4 to A-81;P-5 to A-81; R-6 to A-81; S-7 to A-81; L-8 to A-81; R-9 to A-81; G-10 toA-81; R-11 to A-81; D-12 to A-81; A-13 to A-81; P-14 to A-81; A-15 toA-81; P-16 to A-81; T-17 to A-81; P-18 to A-81; C-19 to A-81; V-20 toA-81; P-21 to A-81; A-22 to A-81; E-23 to A-81; C-24 to A-81; F-25 toA-81; D-26 to A-81; L-27 to A-81; L-28 to A-81; V-29 to A-81; R-30 toA-81; H-31 to A-81; C-32 to A-81; V-33 to A-81; A-34 to A-81; C-35 toA-81; G-36 to A-81; L-37 to A-81; L-38 to A-81; R-39 to A-81; T-40 toA-81; P-41 to A-81; R-42 to A-81; P-43 to A-81; K-44 to A-81; P-45 toA-81; A-46 to A-81; G-47 to A-81; A-48 to A-81; S-49 to A-81; S-50 toA-81; P-51 to A-81; A-52 to A-81; P-53 to A-81; R-54 to A-81; T-55 toA-81; A-56 to A-81; L-57 to A-81; Q-58 to A-81; P-59 to A-81; Q-60 toA-81; E-61 to A-81; S-62 to A-81; V-63 to A-81; G-64 to A-81; A-65 toA-81; G-66 to A-81; A-67 to A-81; G-68 to A-81; E-69 to A-81; A-70 toA-81; A-71 to A-81; L-72 to A-81; P-73 to A-81; L-74 to A-81; P-75 toA-81; and/or G-76 to A-81 of the TR 21 extracellular domain sequenceshown in FIG. 1 (SEQ ID NO:2). Polypeptides encoded by thesepolynucleotides are also encompassed by the invention.

In a most preferred embodiment, the polypeptides of the inventioncomprise, or alternatively consist of amino acids P-16 to L-38 as shownin FIG. 1 (SEQ ID NO:2). Polypeptides at least 90%, at least 95%, atleast 96%, at least 97%, and/or at least 99% identical to amino acidsP-16 to L-38 as shown as FIG. 1 (SEQ ID NO:2) are also encompassed bythe invention. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

In another most preferred embodiment, the polypeptides of the inventioncomprise, or alternatively consist of amino acids P-16 to A-81 as shownin FIG. 1 (SEQ ID NO:2). Polypeptides at least 90%, at least 95%, atleast 96%, at least 97%, and/or at least 99% identical to amino acidsP-16 to A-81 as shown as FIG. 1 (SEQ ID NO:2) are also encompassed bythe invention. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

In another most preferred embodiment, the polypeptides of the inventioncomprise, or alternatively consist of amino acids P-16 to A-83 as shownin FIG. 1 (SEQ ID NO:2). Polypeptides at least 90%, at least 95%, atleast 96%, at least 97%, and/or at least 99% identical to amino acidsP-16 to A-83 as shown as FIG. 1 (SEQ ID NO:2) are also encompassed bythe invention. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, ability to bindTR21 ligands (e.g., Neutrokine alpha) may still be retained). Forexample the ability of the shortened TR21 mutein to induce and/or bindto antibodies which recognize the complete or mature forms of thepolypeptide generally will be retained when less than the majority ofthe residues of the complete or mature polypeptide are removed from theC-terminus. Whether a particular polypeptide lacking C-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a TR21 mutein with a largenumber of deleted C-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six TR21 amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the TR21 polypeptide shown in FIG. 1, up to the arginineresidue at position number 6, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-m¹ of FIG. 1, where m¹is an integer from 6 to 183 corresponding to the position of the aminoacid residue in FIG. 1 (SEQ ID NO:2).

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues: M-1 to Q-183; M-1 to E-182; M-1 to P-181; M-1 toG-180; M-1 to A-179; M-1 to T-178; M-1 to K-177; M-1 to T-176; M-1 toT-175; M-1 to V-174; M-1 to L-173; M-1 to E-172; M-1 to T-171; M-1 toS-170; M-1 to G-169; M-1 to L-168; M-1 to E-167; M-1 to T-166; M-1 toA-165; M-1 to P-164; M-1 to V-163; M-1 to P-162; M-1 to V-161; M-1 toS-160; M-1 to H-159; M-1 to G-158; M-1 to P-157; M-1 to P-156; M-1 toT-155; M-1 to T-154; M-1 to G-153; M-1 to P-152; M-1 to D-151; M-1 toE-150; M-1 to G-149; M-1 to P-148; M-1 to P-147; M-1 to P-146; M-1 toW-145; M-1 to A-144; M-1 to P-143; M-1 to A-142; M-1 to T-141; M-1 toA-140; M-1 to D-139; M-1 to S-138; M-1 to I-137; M-1 to G-136; M-1 toP-135; M-1 to S-134; M-1 to L-133; M-1 to 1-132; M-1 to I-131; M-1 toV-130; M-1 to K-129; M-1 to D-128; M-1 to L-127; M-1 to P-126; M-1 toE-125; M-1 to P-124; M-1 to A-123; M-1 to D-122; M-1 to K-121; M-1 toD-120; M-1 to G-119; M-1 to D-118; M-1 to P-117; M-1 to A-116; M-1 toE-115; M-1 to A-114; M-1 to S-113; M-1 to S-112; M-1 to A-111; M-1 toG-110; M-1 to R-109; M-1 to L-108; M-1 to R-107; M-1 to R-106; M-1 toQ-105; M-1 to R-104; M-1 to R-103; M-1 to R-102; M-1 to W-101; M-1 toS-100; M-1 to V-99; M-1 to L-98; M-1 to G-97; M-1 to V-96; M-1 to L-95;M-1 to V-94; M-1 to L-93; M-1 to A-92; M-1 to L-91; M-1 to V-90; M-1 toL-89; M-1 to A-88; M-1 to L-87; M-1 to G-86; M-1 to L-85; M-1 to L-84;M-1 to A-83; M-1 to P-82; M-1 to A-81; M-1 to G-80; M-1 to G-79; M-1 toL-78; M-1 to L-77; M-1 to G-76; M-1 to P-75; M-1 to L-74; M-1 to P-73;M-1 to L-72; M-1 to A-71; M-1 to A-70; M-1 to E-69; M-1 to G-68; M-1 toA-67; M-1 to G-66; M-1 to A-65; M-1 to G-64; M-1 to V-63; M-1 to S-62;M-1 to E-61; M-1 to Q-60; M-1 to P-59; M-1 to Q-58; M-1 to L-57; M-1 toA-56; M-1 to T-55; M-1 to R-54; M-1 to P-53; M-1 to A-52; M-1 to P-51;M-1 to S-50; M-1 to S-49; M-1 to A-48; M-1 to G-47; M-1 to A-46; M-1 toP-45; M-1 to K-44; M-1 to P-43; M-1 to R-42; M-1 to P-41; M-1 to T-40;M-1 to R-39; M-1 to L-38; M-1 to L-37; M-1 to G-36; M-1 to C-35; M-1 toA-34; M-1 to V-33; M-1 to C-32; M-1 to H-31; M-1 to R-30; M-1 to V-29;M-1 to L-28; M-1 to L-27; M-1 to D-26; M-1 to F-25; M-1 to C-24; M-1 toE-23; M-1 to A-22; M-1 to P-21; M-1 to V-20; M-1 to C-19; M-1 to P-18;M-1 to T-17; M-1 to P-16; M-1 to A-15; M-1 to P-14; M-1 to A-13; M-1 toD-12 M-1 to R-11; M-1 to G-10; M-1 to R-9; M-1 to L-8; M-1 to S-7;and/or M-1 to R-6 of the TR21 sequence disclosed as SEQ ID NO:2 andshown in FIG. 1. Polypeptides encoded by these polynucleotides are alsoencompassed by the invention.

The invention also provides polynucleotides encoding polypeptides havingone or more amino acids deleted from both the amino and the carboxyltermini, which may be described generally as having residues n¹-m¹and/or n²-m¹ of FIG. 1 (i.e., SEQ ID NO:2), where n¹, n², and m¹ areintegers as described above. Thus, any of the above listed N- orC-terminal deletions can be combined to produce a polynucleotideencoding an N- and C-terminal deleted TR21 polypeptide.

In a most preferred embodiment, the polypeptides of the inventioncomprise, or alternatively consist of amino acids P-16 to L-38, or P-16to A-81, P-16 to A-84, as shown in FIG. 1 (SEQ ID NO:2). Polypeptides atleast 90%, at least 95%, at least 96%, at least 97%, and/or at least 99%identical to amino acids P-16 to L-38, P-16 to A-81, P-16 to A-84, asshown in FIG. 1 (SEQ ID NO:2) are also encompassed by the invention.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

The present invention encompasses TR21 polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of FIG. 1 (SEQ ID NO:2), or an epitope of apolypeptide sequence encoded by a polynucleotide that hybridizes to thecomplement of the sequence of SEQ ID NO:1 (e.g., under stringenthybridization conditions or lower stringency hybridization conditions asdefined herein). The present invention further encompassespolynucleotide sequences encoding an epitope of a TR21 polypeptidesequence of the invention (such as, for example, the sequence disclosedin SEQ ID NO:2), polynucleotide sequences of the complementary strand ofa polynucleotide sequence encoding an epitope of the invention, andpolynucleotide sequences which hybridize to this complementary strand(e.g., under stringent hybridization conditions or lower stringencyhybridization conditions defined herein).

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope,” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed herein. (See, e.g., Geysen et al., Proc. Natl. Acad. Sci. USA81:3998-4002 (1983)). Further still, U.S. Pat. No. 5,194,392 to Geysen(1990) describes a general method of detecting or determining thesequence of monomers (amino acids or other compounds) which is atopological equivalent of the epitope (i.e., a “mimotope”) which iscomplementary to a particular paratope (antigen binding site) of anantibody of interest. More generally, U.S. Pat. No. 4,433,092 to Geysen(1989) describes a method of detecting or determining a sequence ofmonomers which is a topographical equivalent of a ligand which iscomplementary to the ligand-binding site of a particular receptor ofinterest. Similarly, U.S. Pat. No. 5,480,971 to Houghten, R. A. et al.(1996) on Peralkylated Oligopeptide Mixtures discloses linearC1-C7-alkyl peralkylated oligopeptides and sets and libraries of suchpeptides, as well as methods for using such oligopeptide sets andlibraries for determining the sequence of a peralkylated oligopeptidethat preferentially binds to an acceptor molecule of interest. Thus,non-peptide analogs of the epitope-bearing peptides of the inventionalso can be made routinely by these methods. Antibodies thatspecifically bind TR21 are also encompassed by the invention.

The term “antigenic epitope,” as used herein, is defined as a portion ofa protein to which an antibody can immunospecifically bind its antigenas determined by any method well known in the art, for example, by theimmunoassays described herein. Immunospecific binding excludesnon-specific binding but does not necessarily exclude cross-reactivitywith other antigens. Antigenic epitopes need not necessarily beimmunogenic.

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 3, at least 4, at least 5, at least 6, at least 7,more preferably at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15, at least 20, at least25, at least 30, at least 40, at least 50, and, most preferably, betweenabout 15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length.

Non-limiting examples of antigenic polypeptides of the invention includeone, two, three, four, five, or more members selected from the group: apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Arg-3 to about Ala-15 in FIG. 1 (SEQ ID NO:2); apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Arg-39 to about Pro-45 in FIG. 1 (SEQ ID NO:2); apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Pro-51 to about Pro-53 in FIG. 1 (SEQ ID NO:2); apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Pro-59 to Glu-61 in FIG. 1 (SEQ ID NO:2); apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Arg-104 to about Ser-112 in FIG. 1 (SEQ ID NO:2); apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Glu-115 to about Asp-122 in FIG. 1 (SEQ ID NO:2); apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Gly-136 to about Ser-138 in FIG. 1 (SEQ ID NO:2); apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Pro-147 to about Gly-153 in FIG. 1 (SEQ ID NO:2);and a polypeptide comprising, or alternatively consisting of, amino acidresidues from about Thr-178 to Glu-182 in FIG. 1 (SEQ ID NO:2). In thiscontext, “about” means the particularly recited ranges and ranges thatare larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acidresidues at either or both the amino- and carboxy-termini. Thesepolypeptide fragments have been determined to bear antigenic epitopes ofthe TR21 polypeptide by the analysis of the Jameson-Wolf antigenicindex, as shown in FIG. 1 and Table I, above. Additional non-exclusivepreferred antigenic epitopes include the antigenic epitopes disclosedherein, as well as portions thereof. Antigenic epitopes are useful, forexample, to raise antibodies, including monoclonal antibodies thatspecifically bind the epitope. Preferred antigenic epitopes include theantigenic epitopes disclosed herein, as well as any combination of two,three, four, five or more of these antigenic epitopes. Antigenicepitopes can be used as the target molecules in immunoassays. (See,e.g., Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science219:660-666 (1983)). Polynucleotides encoding these polypeptides areencompassed by the invention. Additionally, antibodies that bind to oneor more of these polypeptides are also encompassed by the invention.

Additional polypeptides of the invention include one, two, three, four,five, six, seven or more members selected from the group consisting of,amino acids 3 to 15 in SEQ ID NO:2; amino acids 39 to 45 in SEQ ID NO:2;amino acids 51 to 53 in SEQ ID NO:2; amino acids 59 to 61 in SEQ IDNO:2; amino acids 104 to 112 in SEQ ID NO:2; amino acids 136 to 138 inSEQ ID NO:2; amino acids 147 to 153 in SEQ ID NO:2; and amino acids 178to 182 in SEQ ID NO:2. Polynucleotides encoding these polypeptides areencompassed by the invention. Additionally, antibodies that bind to oneor more of these polypeptides are also encompassed by the invention.

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five, six, seven or more of these immunogenicepitopes. The polypeptides comprising one or more immunogenic epitopesmay be presented for eliciting an antibody response together with acarrier protein, such as an albumin, to an animal system (such as rabbitor mouse), or, if the polypeptide is of sufficient length (at leastabout 25 amino acids), the polypeptide may be presented without acarrier. However, immunogenic epitopes comprising as few as 8 to 10amino acids have been shown to be sufficient to raise antibodies capableof binding to, at the very least, linear epitopes in a denaturedpolypeptide (e.g., in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpetHemocyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg of peptide or carrier protein and Freund's adjuvant or anyother adjuvant known for stimulating an immune response. Several boosterinjections may be needed, for instance, at intervals of about two weeks,to provide a useful titer of anti-peptide antibody which can bedetected, for example, by ELISA assay using free peptide adsorbed to asolid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to other polypeptide sequences. Forexample, the polypeptides of the present invention may be fused with theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portionsthereof), or albumin (including, but not limited to, recombinant humanalbumin and fragments or variants thereof (see, e.g., U.S. Pat. No.5,876,969, EP Patent 0413622, and U.S. Pat. No. 5,766,883, hereinincorporated by reference in their entirety). Such fusion proteins mayfacilitate purification and may increase half-life in vivo. This hasbeen shown for chimeric proteins consisting of the first two domains ofthe human CD4-polypeptide and various domains of the constant regions ofthe heavy or light chains of mammalian immunoglobulins. See, e.g., EP394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced deliveryof an antigen across the epithelial barrier to the immune system hasbeen demonstrated for antigens (e.g., insulin) conjugated to an FcRnbinding partner such as IgG or Fc fragments (see, e.g., PCT PublicationsWO 96/22024 and WO 99/04813). IgG Fusion proteins that have adisulfide-linked dimeric structure due to the IgG portion disulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., Proc. Natl. Acad. Sci. USA 88:8972-897(1991)). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix-binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through thetechniques of gene shuffling, motif shuffling, exon shuffling and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of TR21 polynucleotides corresponding to FIG.1 (SEQ ID NO:1) and the polypeptides encoded by these polynucleotidesmay be achieved by DNA shuffling. DNA shuffling involves the assembly oftwo or more DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In another embodimentpolynucleotides of the invention, or polypeptides encoded thereby, maybe altered by random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of a polynucleotide encoding a polypeptide of theinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules.

It will be recognized in the art that some amino acid sequences of TR21can be varied without significant effect on the structure or function ofthe protein. If such differences in sequence are contemplated, it shouldbe remembered that there will be critical areas on the protein whichdetermine activity. Thus, the invention further includes variations ofthe TR21 receptor, which show substantial TR21 receptor activity orwhich include regions of TR21 proteins, such as the protein portionsdiscussed herein. Such mutants include deletions, insertions,inversions, repeats, and type substitutions. As indicated above,guidance concerning which amino acid changes are likely to bephenotypically silent can be found in J. U. Bowie et al., Science247:1306-1310 (1990).

Thus, the fragment, derivative, or analog of the polypeptide of FIG. 1(SEQ ID NO:2), may be (i) one in which at least one or more of the aminoacid residues are substituted with a conserved or non-conserved aminoacid residue (preferably a conserved amino acid residue(s), and morepreferably at least one but less than ten conserved amino acid residues)and such substituted amino acid residue may or may not be one encoded bythe genetic code, or (ii) one in which one or more of the amino acidresidues includes a substituent group, or (iii) one in which the maturepolypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused tothe mature polypeptide, such as an IgG Fc fusion region peptide orleader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the TR21 receptor protein. The preventionof aggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361:266-268(1993), describes certain mutations resulting in selective binding ofTNF-α, to only one of the two known types of TNF receptors. Thus, theTR21 polypeptide receptors of the present invention may include one ormore amino acid substitutions, deletions, or additions, either fromnatural mutations or human manipulation.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table II).

TABLE II Conservative Amino Acid Substitutions Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

In specific embodiments, the number of substitutions, additions ordeletions in the amino acid sequence of FIG. 1 and/or any of thepolypeptide fragments described herein (e.g., the cysteine rich motif,the extracellular domain and/or intracellular domain) is 75, 70, 60, 50,40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 30-20, 20-15,20-10, 15-10, 10-1, 5-10, 1-5, 1-3 or 1-2.

In another embodiment, site directed changes at the amino acid level ofTR21 can be made by replacing a particular amino acid with aconservative substitution. Preferred conservative substitution mutationsof the TR21 amino acid sequence provided in SEQ ID NO:2 include: M1replaced with A, G, I, L, S, T, or V; R2 replaced with H, or K; R3replaced with H, or K; G4 replaced with A, I, L, S, T, M, or V; R6replaced with H, or K; S7 replaced with A, G, I, L, T, M, or V; L8replaced with A, G, I, S, T, M, or V; R9 replaced with H, or K; G10replaced with A, I, L, S, T, M, or V; R11 replaced with H, or K; D12replaced with E; A13 replaced with G, I, L, S, T, M, or V; A15 replacedwith G, I, L, S, T, M, or V; T17 replaced with A, G, I, L, S, M, or V;V20 replaced with A, G, I, L, S, T, or M; A22 replaced with G, I, L, S,T, M, or V; E23 replaced with D; F25 replaced with W, or Y; D26 replacedwith E; L27 replaced with A, G, I, S, T, M, or V; L28 replaced with A,G, I, S, T, M, or V; V29 replaced with A, G, I, L, S, T, or M; R30replaced with H, or K; H31 replaced with K, or R; V33 replaced with A,G, I, L, S, T, or M; A34 replaced with G, I, L, S, T, M, or V; G36replaced with A, I, L, S, T, M, or V; L37 replaced with A, G, I, S, T,M, or V; L38 replaced with A, G, I, S, T, M, or V; R39 replaced with H,or K; T40 replaced with A, G, I, L, S, M, or V; R42 replaced with H, orK; K44 replaced with H, or R; A46 replaced with G, I, L, S, T, M, or V;G47 replaced with A, I, L, S, T, M, or V; A48 replaced with G, I, L, S,T, M, or V; S49 replaced with A, G, I, L, T, M, or V; S50 replaced withA, G, I, L, T, M, or V; A52 replaced with G, I, L, S, T, M, or V; R54replaced with H, or K; T55 replaced with A, G, I, L, S, M, or V; A56replaced with G, I, L, S, T, M, or V; L57 replaced with A, G, I, S, T,M, or V; Q58 replaced with N; Q60 replaced with N; E61 replaced with D;S62 replaced with A, G, I, L, T, M, or V; V63 replaced with A, G, I, L,S, T, or M; G64 replaced with A, I, L, S, T, M, or V; A65 replaced withG, I, L, S, T, M, or V; G66 replaced with A, I, L, S, T, M, or V; A67replaced with G, I, L, S, T, M, or V; G68 replaced with A, I, L, S, T,M, or V; E69 replaced with D; A70 replaced with G, I, L, S, T, M, or V;A71 replaced with G, I, L, S, T, M, or V; L72 replaced with A, G, I, S,T, M, or V; L74 replaced with A, G, I, S, T, M, or V; G76 replaced withA, I, L, S, T, M, or V; L77 replaced with A, G, I, S, T, M, or V; L78replaced with A, G, I, S, T, M, or V; F79 replaced with W, or Y; G80replaced with A, I, L, S, T, M, or V; A81 replaced with G, I, L, S, T,M, or V; A83 replaced with G, I, L, S, T, M, or V; L84 replaced with A,G, I, S, T, M, or V; L85 replaced with A, G, I, S, T, M, or V; G86replaced with A, I, L, S, T, M, or V; L87 replaced with A, G, I, S, T,M, or V; A88 replaced with G, I, L, S, T, M, or V; L89 replaced with A,G, I, S, T, M, or V; V90 replaced with A, G, I, L, S, T, or M; L91replaced with A, G, I, S, T, M, or V; A92 replaced with G, I, L, S, T,M, or V; L93 replaced with A, G, I, S, T, M, or V; V94 replaced with A,G, I, L, S, T, or M; L95 replaced with A, G, I, S, T, M, or V; V96replaced with A, G, I, L, S, T, or M; G97 replaced with A, I, L, S, T,M, or V; L98 replaced with A, G, I, S, T, M, or V; V99 replaced with A,G, I, L, S, T, or M; S100 replaced with A, G, I, L, T, M, or V; W101replaced with F, or Y; R102 replaced with H, or K; R103 replaced with H,or K; R104 replaced with H, or K; Q105 replaced with N; R106 replacedwith H, or K; R107 replaced with H, or K; L108 replaced with A, G, I, S,T, M, or V; R109 replaced with H, or K; G110 replaced with A, I, L, S,T, M, or V; A111 replaced with G, I, L, S, T, M, or V; S112 replacedwith A, G, I, L, T, M, or V; S113 replaced with A, G, I, L, T, M, or V;A114 replaced with G, I, L, S, T, M, or V; E115 replaced with D; A116replaced with G, I, L, S, T, M, or V; D118 replaced with E; G119replaced with A, I, L, S, T, M, or V; D120 replaced with E; K121replaced with H, or R; D122 replaced with E; Al23 replaced with G, I, L,S, T, M, or V; E125 replaced with D; L127 replaced with A, G, I, S, T,M, or V; D128 replaced with E; K129 replaced with H, or R; V130 replacedwith A, G, I, L, S, T, or M; 1131 replaced with A, G, L, S, T, M, or V;1132 replaced with A, G, L, S, T, M, or V; L133 replaced with A, G, I,S, T, M, or V; S134 replaced with A, G, I, L, T, M, or V; G136 replacedwith A, I, L, S, T, M, or V; 1137 replaced with A, G, L, S, T, M, or V;S138 replaced with A, G, I, L, T, M, or V; D139 replaced with E; A140replaced with G, I, L, S, T, M, or V; T141 replaced with A, G, I, L, S,M, or V; A142 replaced with G, I, L, S, T, M, or V; A144 replaced withG, I, L, S, T, M, or V; W145 replaced with F, or Y; G149 replaced withA, I, L, S, T, M, or V; E150 replaced with D; D151 replaced with E; G153replaced with A, I, L, S, T, M, or V; T154 replaced with A, G, I, L, S,M, or V; T155 replaced with A, G, I, L, S, M, or V; G158 replaced withA, I, L, S, T, M, or V; H159 replaced with K, or R; S160 replaced withA, G, I, L, T, M, or V; V161 replaced with A, G, I, L, S, T, or M; V163replaced with A, G, I, L, S, T, or M; A165 replaced with G, I, L, S, T,M, or V; T166 replaced with A, G, I, L, S, M, or V; E167 replaced withD; L168 replaced with A, G, I, S, T, M, or V; G169 replaced with A, I,L, S, T, M, or V; S170 replaced with A, G, I, L, T, M, or V; T171replaced with A, G, I, L, S, M, or V; E172 replaced with D; L173replaced with A, G, I, S, T, M, or V; V174 replaced with A, G, I, L, S,T, or M; T175 replaced with A, G, I, L, S, M, or V; T176 replaced withA, G, I, L, S, M, or V; K177 replaced with H, or R; T178 replaced withA, G, I, L, S, M, or V; A179 replaced with G, I, L, S, T, M, or V; G180replaced with A, I, L, S, T, M, or V; E182 replaced with D; Q183replaced with N; and/or Q184 replaced with N. Polynucleotides encodingthese polypeptides are also encompassed by the invention. The resultingTR21 of the invention may be routinely screened for TR21 functionalactivity and/or physical properties (such as, for example, enhanced orreduced stability and/or solubility). Preferably, the resulting proteinsof the invention have an increased and/or a decreased TR21 functionalactivity. More preferably, the resulting TR21 proteins of the inventionhave more than one increased and/or decreased TR21 functional activityand/or physical property.

Amino acids in the TR21 proteins of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro proliferative activity.Sites that are critical for ligand-receptor binding can also bedetermined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids that may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).

In another embodiment, the invention provides for polypeptides havingamino acid sequences containing non-conservative substitutions of theamino acid sequence provided in SEQ ID NO:2. For example,non-conservative substitutions of the TR21 protein sequence provided inSEQ ID NO:2 include: M1 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; R2 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; R3 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; G4 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P5replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; R6 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; S7 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L8 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; R9 replaced with D, E, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G10 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; R9 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; D12 replaced with H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; A13 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; P14 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or C; A15 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; P16 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, or C; T17 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P18replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; C19 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or P; V20 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P21replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; A22 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E23 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C24replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; F25 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, orC; D26 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; L27 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L28replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V29 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; R30 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; H31 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; C32 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V33 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; A34 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; C35 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or P; G36 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; L37 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L38replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R39 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T40 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; P41 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R42 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P43 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K44 replaced with D,E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P45 replaced with D,E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A46 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; G47 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; A48 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; S49 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S50 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P51 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A52replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P53 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R54 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T55 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A56 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; L57 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; Q58 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,F, W, Y, P, or C; P59 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, or C; Q60 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, F, W, Y, P, or C; E61 replaced with H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; S62 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; V63 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;G64 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A65 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; G66 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; A67 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; G68 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;E69 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; A70 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A71 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L72 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; P73 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; L74 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; P75 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, or C; G76 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; L77 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L78replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F79 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G80 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A81 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; P82 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or C; A83 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L84 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L85 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G86 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L87 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; A88 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L89 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V90 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L91 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A92 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; L93 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; V94 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L95 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V96 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; G97 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; L98 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; V99 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S100 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W101 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R102 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R103 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R104 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q105 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R106replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R107replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L108replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R109 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G110 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A111 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; S112 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; S113 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A114replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E115 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A116 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; P117 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; D118 replaced with H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G119 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; D120 replaced with H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K121 replaced with D, E, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D122 replaced with H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Al23 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; P124 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; E125 replaced with H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; P126 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L127 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; D128 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; K129 replaced with D, E, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; V130 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; 1131 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; 1132 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L133replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S134 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; P135 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G136 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; 1137 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; S138 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;D139 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; A140 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T141replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A142 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; P143 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A144 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; W145 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; P146 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; P147 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P148 replaced with D, E, H, K,R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G149 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; E150 replaced with H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; D151 replaced with H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; P152 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G153 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; T154 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; T155 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;P156 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or C; P157 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, or C; G158 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;H159 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;S160 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V161 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; P162 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; V163 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; P164 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A165 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; T166 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; E167 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; L168 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;G169 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S170 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; T171 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; E172 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; L173 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; V174 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; T175 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T176replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K177 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T178 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A179 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; G180 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; P181 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, or C; E182 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; Q183 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, F, W, Y, P, or C; and/or Q184 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C. Polynucleotides encoding thesepolypeptides are also encompassed by the invention. The resulting TR21proteins of the invention may be routinely screened for TR21 functionalactivities and/or physical properties (such as, for example, enhanced orreduced stability and/or solubility) described throughout thespecification and known in the art. Preferably, the resulting proteinsof the invention have an increased and/or a decreased TR21 functionalactivity. More preferably, the resulting TR21 proteins of the inventionhave more than one increased and/or decreased TR21 functional activityand/or physical property.

To improve or alter the characteristics of TR21 polypeptides, proteinengineering may be employed. Recombinant DNA technology known to thoseskilled in the art can be used to create novel mutant proteins or“muteins including single or multiple amino acid substitutions,deletions, additions or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions.

Non-naturally occurring variants may be produced using art-knownmutagenesis techniques, which include, but are not limited tooligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis,site directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res.13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)),cassette mutagenesis (see e.g., Wells et al., Gene 34:315 (1985)),restriction selection m utagenesis (see e.g., Wells et al., Philos.Trans. R. Soc. London SerA 317:415 (1986)).

Thus, the invention also encompasses TR21 derivatives and analogs thathave one or more amino acid residues deleted, added, or substituted togenerate TR21 polypeptides that are better suited for expression, scaleup, etc., in the host cells chosen. For example, cysteine residues canbe deleted or substituted with another amino acid residue in order toeliminate disulfide bridges; N-linked glycosylation sites can be alteredor eliminated to achieve, for example, expression of a homogeneousproduct that is more easily recovered and purified from yeast hostswhich are known to hyperglycosylate N-linked sites. To this end, avariety of amino acid substitutions at one or both of the first or thirdamino acid positions on any one or more of the glycosylationrecognitions sequences in the TR21 polypeptides of the invention, and/oran amino acid deletion at the second position of any one or more suchrecognition sequences will prevent glycosylation of the TR21 at themodified tripeptide sequence (see, e.g., Miyajimo et al., EMBO J5(6):1193-1197). Additionally, one or more of the amino acid residues ofthe polypeptides of the invention (e.g., arginine and lysine residues)may be deleted or substituted with another residue to eliminateundesired processing by proteases such as, for example, furins orkexins.

The polypeptides of the present invention include a polypeptidecomprising, or alternatively, consisting of: amino acids 1 to 184 inFIG. 1 (SEQ ID NO:2); amino acids 2 to 184 in FIG. 1 (SEQ ID NO:2); theTR21 extracellular domain; the TR21 cysteine rich motif; the TR21transmembrane domain; the intracellular domain of TR21; and the TR21extracellular domain and the TR21 intracellular domain with all or partof the transmembrane domain deleted; as well as polypeptides which areat least 80% identical, more preferably at least 90% or 95% identical,still more preferably at least 96%, 97%, 98%, 99% or 100% identical tothe polypeptides described above (e.g., the polypeptide of FIG. 1 (SEQID NO:2)), and also include portions of such polypeptides with at least30 amino acids and more preferably at least 50 or at least 100 aminoacids. Polynucleotides encoding these polypeptides are also encompassedby the invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a TR21 polypeptideis intended that the amino acid sequence of the polypeptide is identicalto the reference sequence except that the polypeptide sequence mayinclude up to five amino acid alterations per each 100 amino acids ofthe reference amino acid of the TR21 receptor. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to areference amino acid sequence, up to 5% of the amino acid residues inthe reference sequence may be deleted or substituted with another aminoacid, or a number of amino acids up to 5% of the total amino acidresidues in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to, for instance,the amino acid sequence shown in FIG. 1 (SEQ ID NO:2), can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter. According to this embodiment, if the subject sequence isshorter than the query sequence due to N- or C-terminal deletions, notbecause of internal deletions, a manual correction is made to theresults to take into consideration the fact that the FASTDB program doesnot account for N- and C-terminal truncations of the subject sequencewhen calculating global percent identity. For subject sequencestruncated at the N- and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminal of the subject sequence,which are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. A determination ofwhether a residue is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thisfinal percent identity score is what is used for the purposes of thisembodiment. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence. For example, a 90 aminoacid residue subject sequence is aligned with a 100 residue querysequence to determine percent identity. The deletion occurs at theN-terminus of the subject sequence and therefore, the FASTDB alignmentdoes not show a matching/alignment of the first 10 residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.In another example, a 90 residue subject sequence is compared with a 100residue query sequence. This time the deletions are internal deletionsso there are no residues at the N- or C-termini of the subject sequencewhich are not matched/aligned with the query. In this case the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N- and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected for. Noother manual corrections are made for the purposes of this embodiment.

In additional embodiments, polynucleotides of the invention comprise, oralternatively consist of, a polynucleotide sequence at least 80%, 85%,90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to thepolynucleotide sequence encoding the extracellular cysteine rich motifof TR21 disclosed in FIG. 1 (amino acid residues from 19 to 35). Inanother embodiment, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide that hybridizes under stringenthybridization conditions to DNA complementary to the polynucleotidesequence encoding the TR21 extracellular cysteine rich motif. Thepresent invention also encompasses the above polynucleotide/nucleic acidsequences fused to a heterologous polynucleotide sequence. Polypeptidesencoded by these nucleic acids and/or polynucleotide sequences are alsoencompassed by the invention.

The present application is also directed to proteins containingpolypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%identical to the TR21 polypeptide sequence set forth as n¹-m¹, and/orn²-m¹ as described herein. In preferred embodiments, the application isdirected to proteins comprising or alternatively consisting of,polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%identical to polypeptides having the amino acid sequence of the specificTR21 N- and C-terminal deletions recited herein. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

In certain preferred embodiments, TR21 proteins of the inventioncomprise fusion proteins as described above wherein the TR21polypeptides are those described as n¹-m¹, and/or n²-m¹ as describedherein. In preferred embodiments, the application is directed to nucleicacid molecules at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%identical to the nucleic acid sequences encoding polypeptides having theamino acid sequence of the specific N- and C-terminal deletions recitedherein. Polynucleotides encoding these polypeptides are also encompassedby the invention.

In preferred embodiments, the TR21 polypeptide or fragment thereof bindsa Neutrokine-alpha polypeptide or splice variants thereof. The abilityof a TR21 polypeptide (e.g., fragment) of the invention to bind aNeutrokine-alpha polypeptide, or a splice variant thereof, can routinelybe determined using techniques described herein or otherwise known inthe art. In another non-exclusive preferred embodiment, a TR21polypeptide, or fragment thereof, of the invention antagonizes B cellproliferation and/or differentiation merdiated by Neutokine-alpha or asplice variant thereof. The ability of a TR21 polypeptide to antagonizeB cell proliferation and/or differentiation mediated by Neutrokine-alphaor a splice variant thereof can routinely be determined using techniquesdescribed herein or otherwise known in the art.

In preferred embodiments, the TR21 polypeptide or fragment thereof bindsa heteromultimeric polypeptide complex comprising one or moreNeutrokine-alpha polypeptides or splice avariants thereof. The abilityof a TR21 polypeptide (e.g., fragment) of the invention to bind aheteromultimeric polypeptide complex comprising one or more Neutrokinealpha polypeptides or splice variants thereof can routinely bedetermined using techniques described herein or otherwise known in theart. In another non-exclusive preferred embodiment, a TR21 polypeptideor fragment thereof of the invention antagonizes B cell proliferationand/or differentiation mediated by a heteromultimeric polypeptidecomplex comprising one or more Neutrokine-alpha polypeptides or splicevariants thereof. The ability of a TR21 polypeptide to antagonize B cellproliferation and/or differentiation mediated by a heteromultimericpolypeptide complex comprising one or more Neutrokine-alpha polypeptidesor splice variants thereof can routinely be determined using techniquesdescribed herein or otherwise known in the art.

In one embodiment, one or more of the TR21 polypeptides of the inventionare expressed at relatively high levels in mature B cells. In a specificembodiment, expression of one or more of the TR21 polypeptides of theinvention is restricted to mature B cells.

In one embodiment, the B cell proliferation assay described herein maybe modified for use in screening for TR21 related proteins or agonistsor antagonists thereof. In this instance, a baseline level of B cellproliferation and/or differentiation mediated by Neutrokine-alpha orNeutrokine-alpha SV is determined. Potential TR21 related proteins orpolypeptide(s) are added to an experimental well and the resultant levelof B cell proliferation and/or differentiation mediated byNeutrokine-alpha or Neutrokine-alpha SV is assessed and compared to thebaseline level. An increase in B cell proliferation and/ordifferentiation in the experimental well will indicate that thepotential TR21 related proteins(s) or polypeptide(s) is either (or both)a TR21 related protein or an agonist. Decreased B cell proliferationand/or differentiation will indicate that the potential TR21 protein(s)or polypeptide(s) is an antagonist.

In another embodiment, the B cell proliferation assay may be modifiedfor use in screening for TR21 related proteins or agonists orantagonists thereof using heteromultimeric polypeptide complexescomprising one or more Neutrokine-alpha or Neutrokine-alpha SV proteins.The baseline level of B cell proliferation and/or differentiationmediated by a heteromultimeric polypeptide complex comprising one ormore Neutrokine-alpha or Neutrokine-alpha SV proteins may be determinedby routinely modifying the techniques described herein. Potential TR21related proteins or polypeptide(s) are added to an experimental well andthe resultant level of B cell proliferation and/or differentiation isassessed and compared to the baseline level. An increase in B cellproliferation and/or differentiation in the experimental well willindicate that the potential TR21 related proteins(s) or polypeptide(s)is either (or both) a TR21 related protein or an agonist. Decreased Bcell proliferation and/or differentiation will indicate that thepotential TR21 protein(s) or polypeptide(s) is an antagonist

In another embodiment, TR21 polypeptides (including TR21 solublefragments) may be identified by means of a functional screen using themodified B cell proliferation assay as described above.

Moreover, the TR21 polypeptides described herein may be used as a meansof detecting and/or quantifying levels of Neutrokine-alpha orNeutrokine-alpha SV or heteromultimeric polypeptide complexes comprisingNeutrokine-alpha or Neutrokine-alpha SV in a sample (e.g., a biologicalsample) by for example using, or routinely modifying immunoassays knownin the art, and/or using or routinely modifying, the B cellproliferation assay described herein.

TR21 Antibodies

Further polypeptides of the invention relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a TR21polypeptide, polypeptide fragment, or variant of SEQ ID NO:2, and/or aTR21 epitope (as determined by immunoassays well known in the art forassaying specific antibody-antigen binding). Antibodies of the inventioninclude, but are not limited to, polyclonal, monoclonal, multispecific,human, humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′) fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Idantibodies to antibodies of the invention), and epitope-bindingfragments of any of the above. The term “antibody,” as used herein,refers to immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain anantigen-binding site that immunospecifically binds an antigen. Theimmunoglobulin molecules of the invention can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2) or subclass of immunoglobulin molecule. In specificembodiments, the immunoglobulin molecules of the invention are IgG1. Inother specific embodiments, the immunoglobulin molecules of theinvention are IgG4.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, rat and/or rabbit homologs of human proteinsand the corresponding epitopes thereof. Antibodies that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M,5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M or 10⁻⁸ M. Even morepreferred binding affinities include those with a dissociation constantor Kd less than 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹M, 5×10⁻¹² M, ¹⁰⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵M, or 10⁻¹⁵ M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. Preferably, antibodies of the present invention bind an antigenicepitope disclosed herein (e.g., amino acid residues 3 to 15, 39 to 45,51 to 53, 59 to 61, 104 to 112, 115 to 122, 136 to 138, 147 to 153 and178 to 182 of SEQ ID NO:2), or a portion thereof. The invention featuresboth receptor-specific antibodies and ligand-specific antibodies. Theinvention also features receptor-specific antibodies that do not preventligand binding but prevent receptor activation. Receptor activation(i.e., signaling) may be determined by techniques described herein orotherwise known in the art. For example, receptor activation can bedetermined by detecting activation of the transcription factors NF-AT,AP-1, and/or NF-KAPPAB using techniques known in the art, and/or thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand activity or receptor activity by atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, or at least 50% of the activity in absence ofthe antibody. In other specific embodiments, antibodies are providedthat promote ligand activity or receptor activity by at least 95%, atleast 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing multimerization and/or aggregation (i.e., via antibodycross-linking) of the receptor (i.e., TR21). The antibodies may bespecified as agonists, antagonists or inverse agonists for biologicalactivities comprising the specific biological activities of the peptidesof the invention disclosed herein. The above antibody agonists can bemade using methods known in the art. See, e.g., PCT publication WO96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988(1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al.,J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res.58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179(1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard etal., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al.,Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem.272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995);Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al.,Cytokine 8(1):14-20 (1996) (which are all incorporated by referenceherein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

By way of another non-limiting example, antibodies of the invention maybe administered to individuals as a form of passive immunization.Alternatively, antibodies of the present invention may be used forepitope mapping to identify the epitope(s) bound by the antibody.Epitopes identified in this way may, in turn, for example, be used asvaccine candidates, i.e., to immunize an individual to elicit antibodiesagainst the naturally occurring forms of TR21.

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples (e.g., Example 3). In a non-limitingexample, mice can be immunized with a polypeptide of the invention or acell expressing such peptide. Once an immune response is detected, e.g.,antibodies specific for the antigen are detected in the mouse serum, themouse spleen is harvested and splenocytes isolated. The splenocytes arethen fused by well-known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC™. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies can begenerated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Protocols for generating EBV-transformed B cell lines are commonly knownin the art, such as, for example, the protocol outlined in Chapter 7.22of Current Protocols in Immunology, Coligan et al., Eds., 1994, JohnWiley & Sons, NY, which is hereby incorporated in its entirety byreference herein. The source of B cells for transformation is commonlyhuman peripheral blood, but B cells for transformation may also bederived from other sources including, but not limited to, lymph nodes,tonsil, spleen, tumor tissue, and infected tissues. Tissues aregenerally made into single cell suspensions prior to EBV transformation.Investigators may also choose to perform selection procedures that willenrich the sample for B cells that are antigen-reactive. For example,one method of enriching for antigen-reactive B cells is panning on aplastic dish that has been coated with antigen. Antigen reactive B cellsmay then be eluted from the plastic dish and used for transformation.Alternatively, it is possible to enrich for antigen-reactive B cellsusing fluorescence activated cells sorting (FACS). In this method, onemight use fluorescently labeled antigen to sort out a population ofantigen reactive B-cells from non-antigen reactive B cells an othercells types. Both FACS analysis and panning, may also be performed in amanner so as to enrich for B cells as opposed to antigen-reactive Bcells. The advantage of selecting for total B cells populations is thatone is more likely to include plasma cells, or B cells activelysecreting immunoglobulin, that might be missed in procedures thatrequire the presence of cell-surface immunoglobulin for detection.Growth of EBV-infected cells is promoted by monocytes, so investigatorsmay wish to take care not to exclude these form culture, or to resupplymonocytes after selection procedures. Additionally, steps may be takento either physically remove or inactivate T cells (e.g., by treatmentwith cyclosporin A) in B cell-containing samples, because T cells fromindividuals seropositive for anti-EBV antibodies can suppress B cellimmortalization by EBV.

In general, the sample containing human B cells is innoculated with EBV,and cultured for 3-4 weeks. A typical source of EBV is the culturesupernatant of the B95-8 cell line (ATCC™ #VR-1492). Physical signs ofEBV transformation can generally be seen towards the end of the 3-4 weekculture period. By phase-contrast microscopy, transformed cells mayappear large, clear, hairy and tend to aggregate in tight clusters ofcells. Initially, EBV lines are generally polyclonal. However, overprolonged periods of cell cultures, EBV lines may become monoclonal orpolyclonal as a result of the selective outgrowth of particular B cellclones. Alternatively, polyclonal EBV transformed lines may be subcloned(e.g., by limiting dilution culture) or fused with a suitable fusionpartner and plated at limiting dilution to obtain monoclonal B celllines. Suitable fusion partners for EBV transformed cell lines includemouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma celllines (human×mouse; e.g., SPAM-8, SBC-H20, and CB-F7), and human celllines (e.g., GM 1500, SKO-007, RPMI 8226, and KR-4). Thus, the presentinvention also provides a method of generating polyclonal or monoclonalhuman antibodies against polypeptides of the invention or fragmentsthereof, comprising EBV-transformation of human B cells.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles that carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen-binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phages used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entirety. Humanized antibodies are antibodymolecules from non-human species antibody that binds the desired antigenhaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework regions from a human immunoglobulinmolecule. Often, framework residues in the human framework regions willbe substituted with the corresponding residue from the CDR donorantibody to alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Pathan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; 5,939,598; 6,075,181; and 6,114,598, which areincorporated by reference herein in their entirety. In addition,companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (SanJose, Calif.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8): 2429-2438 (1991)).For example, antibodies which bind to and competitively inhibitpolypeptide multimerization and/or binding of a polypeptide of theinvention to a ligand can be used to generate anti-idiotypes that“mimic” the polypeptide multimerization and/or binding domain and, as aconsequence, bind to and neutralize polypeptide and/or its ligand (e.g.,Neutrokine-alpha). Such neutralizing anti-idiotypes or Fab fragments ofsuch anti-idiotypes can be used in therapeutic regimens to neutralizepolypeptide ligand. For example, such anti-idiotypic antibodies can beused to bind a polypeptide of the invention and/or to bind itsligands/receptors, and thereby block its biological activity.

Intrabodies of the invention can be produced using methods known in theart, such as those disclosed and reviewed in Chen et al., Hum. GeneTher. 5:595-601 (1994); Marasco, W. A., Gene Ther. 4:11-15 (1997);Rondon and Marasco, Annu. Rev. Microbiol. 51:257-283 (1997); Proba etal., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128(1999); Ohage et al., J. Mol. Biol. 291:1129-1134 (1999); Wirtz andSteipe, Protein Sci. 8:2245-2250 (1999); Zhu et al., J. Immunol. Methods231:207-222 (1999); and references cited therein. In particular, a CCR5intrabody has been produced by Steinberger et al., Proc. Natl. Acad.Sci. USA 97:805-810 (2000).

Polynucleotides Encoding TR21 Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a TR21 polypeptide of the invention, preferably,an antibody that binds to a polypeptide having the amino acid sequenceof SEQ ID NO:2.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(See, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

Methods of Producing TR21 Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques. Methodsof producing antibodies include, but are not limited to, hybridomatechnology, EBV transformation, and other methods discussed herein aswell as through the use recombinant DNA technology, as discussed below.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (See e.g., Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. An advantage of glutaminesynthase based vectors are the availability of cell lines (e.g., themurine myeloma cell line, NS0) which are glutamine synthase negative.Glutamine synthase expression systems can also function in glutaminesynthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) byproviding additional inhibitor to prevent the functioning of theendogenous gene. A glutamine synthase expression system and componentsthereof are detailed in PCT publications: WO87/04462; WO86/05807;WO89/01036; WO89/10404; and WO91/06657 which are incorporated in theirentireties by reference herein. Additionally, glutamine synthaseexpression vectors that may be used according to the present inventionare commercially available from suppliers, including, for example LonzaBiologics, Inc. (Portsmouth, N.H.). Expression and production ofmonoclonal antibodies using a GS expression system in murine myelomacells is described in Bebbington et al., Bio/technology 10:169(1992) andin Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which areincorporated in their entireties by reference herein.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressingboth heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion or a portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms may be made by fusing polypeptides to portionsof IgA and IgM. Methods for fusing or conjugating the polypeptides ofthe present invention to antibody portions are known in the art. See,e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053;5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995);and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341(1992) (saidreferences incorporated by reference in their entireties).

As discussed, supra, the polypeptides corresponding to a TR21polypeptide, polypeptide fragment, or a variant of SEQ ID NO:2 may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides corresponding to SEQID NO:2 may be fused or conjugated to the above antibody portions tofacilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP 394,827; Traunecker etal., Nature 331:84-86 (1988). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson etal., J. Biol. Chem. 270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “Flag™” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude iodine (¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹¹In, ¹¹²In, ^(113m)In, ^(115m)In), technetium(⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu,¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr,¹⁰⁵Rh, and ⁹⁷Ru.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, ²¹³Bi, or other radioisotopes suchas, for example, ²¹¹At, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ¹²⁵I, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn,⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm , ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Ct, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, ⁹⁰Y,¹¹⁷Tin, ¹⁸⁶Re, ¹⁸⁸Re and ¹⁶⁶Ho. In specific embodiments, an antibody orfragment thereof is attached to macrocyclic chelators useful forconjugating radiometal ions, including but not limited to, ¹⁷⁷ _(Lu,)⁹⁰Y, ¹⁶⁶Ho, ¹¹¹In, and ¹⁵³Sm, to polypeptides. In a preferredembodiment, the radiometal ion associated with the macrocyclic chelatorsattached to TR21 polypeptides of the invention is ¹¹¹In. In anotherpreferred embodiment, the radiometal ion associated with the macrocyclicchelator attached to TR21 polypeptides of the invention is ⁹⁰Y. Inspecific embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inother specific embodiments, the DOTA is attached to an antibody of theinvention or fragment thereof via a linker molecule. Examples of linkermolecules useful for conjugating DOTA to a polypeptide are commonlyknown in the art—see, for example, DeNardo et al., Clin Cancer Res.4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7(1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999) whichare hereby incorporated by reference in their entirety. In addition,U.S. Pat. Nos. 5,652,361 and 5,756,065, which disclose chelating agentsthat may be conjugated to antibodies, and methods for making and usingthem, are hereby incorporated by reference in their entireties.

A cytotoxin or cytotoxic agent includes any agent that is detrimental tocells and includes such molecules as small molecule toxins andenzymatically active toxins of bacterial, fungal, plant, or animalorigin, or fragments thereof. Examples include paclitaxol, cytochalasinB, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide(VP-16), tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)(cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin)and doxorubicin), antibiotics (e.g., dactinomycin (formerlyactinomycin), bleomycin, mithramycin, and anthramycin (AMC)),anti-mitotic agents (e.g., vincristine and vinblastine), improsulfan,piposulfan, benzodopa, carboquone, meturedopa, uredopa, altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide trimethylolomelamine, chlomaphazine,cholophosphamide, estramustine, ifosfamide, novembichin, phenesterine,prednimustine, trofosfamide, uracil mustard, chlorozotocin, fotemustine,nimustine, ranimustine, aclacinomysins, azaserine, cactinomycin,calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins,detorubicin, 6-diazo-5-oxo-L-norleucine, epirubicin, esorubicin,idarubicin, marcellomycin, mycophenolic acid, nogalamycin, olivomycins,peplomycin, potfiromycin, quelamycin, rodorubicin, streptonigrin,tubercidin, ubenimex, zinostatin, zorubicin, denopterin, pteropterin,trimetrexate, fludarabine, thiamiprine, ancitabine, azacitidine,6-azauridine, carmofur, dideoxyuridine, doxifluridine, enocitabine,floxuridine, 5-FU, calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane,frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinicacid, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine,demecolcine, diaziquone, elfornithine, elliptiniurn acetate, etoglucid,gallium nitrate, hydroxyurea, lentinan, lonidamine, mitoguazone,mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, podophyllinicacid, 2-ethylhydrazide, procarbazine, PSKO, razoxane, sizofiran,spirogermanium, tenuazonic acid, triaziquone,2,2′,2″-trichlorotriethylamine, urethan, vindesine, dacarbazine,mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine,arabinoside (“Ara-C”), taxoids, e.g. paclitaxel (TAXOL”, Bristol-MyersSquibb Oncology, Princeton, N.J.) doxetaxel (TAXOTERE”, Rh6ne-PoulencRorer, Antony, France), gemcitabine, ifosfamide, vinorelbine, navelbine,novantrone, teniposide, aminopterin, XELODA™, ibandronate, CPT-I 1,topoisomerase inhibitor RFS 2000, difluoromethylomithine (DMFO),retinoic acid, esperamicins, capecitabine, and pharmaceuticallyacceptable salts, acids or derivatives of any of the above. Alsoincluded in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogensincluding for example tamoxifen, raloxifene, aromatase inhibiting4(5)-imidazoles, 4 hydroxytamoxifen, trioxifene, keoxifene, LY 117018,onapristone, toremifene (Fareston), and anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin, andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), CD40-ligand, a thrombotic agent or an anti-angiogenicagent, e.g., angiostatin or endostatin; or, biological responsemodifiers such as, for example, lymphokines, interleukin-1 (“IL-1”),interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophagecolony stimulating factor (“GM-CSF”), granulocyte colony stimulatingfactor (“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Anton et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Immunophenotyping using TR21 Antibodies

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the geneof the present invention may be useful as a cell specific marker, ormore specifically as a cellular marker that is differentially expressedat various stages of differentiation and/or maturation of particularcell types. Monoclonal antibodies directed against a specific epitope,or combination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Assays for TR21 Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trayslol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzyme (e.g., horseradishperoxidase or alkaline phosphatase) or radioactive molecule (e.g., ³²Por ¹²⁵I) diluted in blocking buffer, washing the membrane in washbuffer, and detecting the presence of the antigen. One of skill in theart would be knowledgeable as to the parameters that can be modified toincrease the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzyme (e.g., horseradishperoxidase or alkaline phosphatase) to the well and incubating for aperiod of time, and detecting the presence of the antigen. In ELISAs theantibody of interest does not have to be conjugated to a detectablecompound; instead, a second antibody (which recognizes the antibody ofinterest) conjugated to a detectable compound may be added to the well.Further, instead of coating the well with the antigen, the antibody maybe coated to the well. In this case, a second antibody conjugated to adetectable compound may be added following the addition of the antigenof interest to the coated well. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thesignal detected as well as other variations of ELISAs known in the art.For further discussion regarding ELISAs see, e.g., Ausubel et al, eds,1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., ³H or ¹²⁵I)in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic uses of TR21 Antibodies

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein.

For example, antibody antagonists of the invention may be used to treat,inhibit or prevent autoimmune diseases, disorders, or conditionsassociated with such diseases or disorders, including, but not limitedto, autoimmune hemolytic anemia (including, but not limited tocryoglobinemia or Coombs positive anemia), autoimmune neonatalthrombocytopenia, idiopathic thrombocytopenia purpura, autoimmuneneutropenia, autoimmunocytopenia, hemolytic anemia, antiphospholipidsyndrome, dermatitis (e.g. atopic dermatitis), gluten-sensitiveenteropathy, allergic encephalomyelitis, myocarditis, relapsingpolychondritis, rheumatic heart disease, Multiple Sclerosis, Neuritis,Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g.,Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome,Autoimmune Pulmonary Inflammation, IgA glomerulonephritis, dense depositdisease, Guillain-Barre Syndrome, diabetes mellitus (e.g. Type Idiabetes mellitus or insulin dependent diabetes mellitis), juvenileonset diabetes, and autoimmune inflammatory eye, autoimmune thyroiditis,hypothyroidism (i.e., Hashimoto's thyroiditis, systemic lupuserhythematosus, discoid lupus, Goodpasture's syndrome, Pemphigus,receptor autoimmunities such as, for example, (a) Graves' Disease, (b)Myasthenia Gravis, and (c) insulin resistance, autoimmunethrombocytopenic purpura, rheumatoid arthritis, scleroderma withanti-collagen antibodies, mixed connective tissue disease,polymyositis/dermatomyositis, pernicious anemia (Addison's disease),idiopathic Addison's disease, infertility, glomerulonephritis such asprimary glomerulonephritis and IgA nephropathy, bullous pemphigoid,Sjogren's syndrome, and adrenergic drug resistance (including adrenergicdrug resistance with asthma or cystic fibrosis), chronic activehepatitis, primary biliary cirrhosis, other endocrine gland failure,vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopicdermatitis, asthma, inflammatory myopathies, and other inflammatory,granulamatous, degenerative, atrophic disorders, and other disorderssuch as inflammatory skin diseases including psoriasis and sclerosis,responses associated with inflammatory bowel disease (such as Crohn'sdisease and ulcerative colitis), respiratory distress syndrome(including adult respiratory distress syndrome, ARDS), meningitis,encephalitis, colitis, allergic conditions such as eczema and otherconditions involving infiltration of T cells and chronic inflammatoryresponses, atherosclerosis, leukocyte adhesion deficiency, Reynaud'ssyndrome, and immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes typically foundin tuberculosis, sarcoidosis, granulomatosis and diseases involvingleukocyte diapedesis, central nervous system (CNS) inflammatorydisorder, multiple organ injury syndrome, antigen-antibody complexmediated diseases, anti-glomerular basement membrane disease,Lambert-Eaton myasthenic syndrome, Beheet disease, giant cell arteritis,immune complex nephritis, IgA nephropathy, IgM polyneuropathies orautoimmune thrombocytopenia etc.

In a specific embodiment, antibodies of the invention are used to treat,inhibit, prognose, diagnose or prevent rheumatoid arthritis. In aspecific embodiment, antibodies of the invention are used to treat,inhibit, prognose, diagnose or prevent advanced rheumatoid arthritis. Inanother specific embodiment, antibodies of the invention are used totreat, inhibit, prognose, diagnose or prevent systemic lupuserythematosis.

For example, an antibody, or antibodies, of the present invention areused to treat patients with clinical diagnosis of rheumatoid arthritis(RA). The patient treated will not have a B cell malignancy. Moreover,the patient is optionally further treated with any one or more agentsemployed for treating RA such as salicylate; nonsteroidalanti-inflammatory drugs such as indomethacin, phenylbutazone,phenylacetic acid derivatives (e.g. ibuprofen and fenoprofen),naphthalene acetic acids (naproxen), pyrrolealkanoic acid (tometin),indoleacetic acids (sulindac), halogenated anthranilic acid(meclofenamate sodium), piroxicam, zomepirac and diflunisal;antimalarials such as chloroquine; gold salts; penicillamine; orimmunosuppressive agents such as methotrexate or corticosteroids indosages known for such drugs or reduced dosages. Preferably however, thepatient is only treated with an antibody, or antibodies, of the presentinvention. Antibodies of the present invention are administered to theRA patient according to a dosing schedule as described infra, which maybe readily determined by one of ordinary skill in the art. The primaryresponse is determined by the Paulus index (Paulus et al. AthritisRheum. 33:477-484 (1990)), i.e. improvement in morning stiffness, numberof painful and inflamed joints, erythrocyte sedimentation (ESR), and atleast a 2-point improvement on a 5-point scale of disease severityassessed by patient and by physician. Administration of an antibody, orantibodies, of the present invention will alleviate one or more of thesymptoms of RA in the patient treated as described above.

In a further specific embodiment, antibodies of the invention are usedto treat, inhibit, prognose, diagnose or prevent hemolytic anemia. Forexample, patients diagnosed with autoimmune hemolytic anemia (AIHA),e.g., cryoglobinemia or Coombs positive anemia, are treated with anantibody, or antibodies, of the present invention. AIHA is an acquiredhemolytic anemia due to auto-antibodies that react with the patient'sred blood cells. The patient treated will not have a B cell malignancy.Further adjunct therapies (such as glucocorticoids, prednisone,azathioprine, cyclophosphamide, vinca-laden platelets or Danazol) may becombined with the antibody therapy, but preferably the patient istreated with an antibody, or antibodies, of the present invention as asingle-agent throughout the course of therapy. Antibodies of the presentinvention are administered to the hemolytic anemia patient according toa dosing schedule as described infra, which may be readily determined byone of ordinary skill in the art. Overall response rate is determinedbased upon an improvement in blood counts, decreased requirement fortransfusions, improved hemoglobin levels and/or a decrease in theevidence of hemolysis as determined by standard chemical parameters.Administration of an antibody, or antibodies of the present inventionwill improve any one or more of the symptoms of hemolytic anemia in thepatient treated as described above. For example, the patient treated asdescribed above will show an increase in hemoglobin and an improvementin chemical parameters of hemolysis or return to normal as measured byserum lactic dehydrogenase and/or bilirubin.

In another specific embodiment, antibodies of the invention are used totreat, inhibit, prognose, diagnose or prevent adult immunethrombocytopenic purpura. Adult immune thrombocytopenic purpura (ITP) isa relatively rare hematologic disorder that constitutes the most commonof the immune-mediated cytopenias. The disease typically presents withsevere thrombocytopenia that may be associated with acute hemorrhage inthe presence of normal to increased megakaryocytes in the bone marrow.Most patients with ITP have an IgG antibody directed against targetantigens on the outer surface of the platelet membrane, resulting inplatelet sequestration in the spleen and accelerated reticuloendothelialdestruction of platelets (Bussell, J. B. Hematol. Oncol. Clin. North Am.(4):179 (1990)). A number of therapeutic interventions have been shownto be effective in the treatment of ITP. Steroids are generallyconsidered first-line therapy, after which most patients are candidatesfor intravenous immunoglobulin (IVIG), splenectomy, or other medicaltherapies including vincristine or immunosuppressive/cytotoxic agents.Up to 80% of patients with ITP initially respond to a course ofsteroids, but far fewer have complete and lasting remissions.Splenectomy has been recommended as standard second-line therapy forsteroid failures, and leads to prolonged remission in nearly 60% ofcases yet may result in reduced immunity to infection. Splenectomy is amajor surgical procedure that may be associated with substantialmorbidity (15%) and mortality (2%). IVIG has also been used as secondline medical therapy, although only a small proportion of adult patientswith ITP achieve remission. Therapeutic options that would interferewith the production of autoantibodies by activated B cells without theassociated morbidities that occur with corticosteroids and/orsplenectomy would provide an important treatment approach for aproportion of patients with ITP. Patients with clinical diagnosis of ITPare treated with an antibody, or antibodies of the present invention,optionally in combination with steroid therapy. The patient treated willnot have a B cell malignancy. Antibodies of the present invention areadministered to the RA patient according to a dosing schedule asdescribed infra, which may be readily determined by one of ordinaryskill in the art. Overall patient response rate is determined based upona platelet count determined on two consecutive occasions two weeks apartfollowing treatments as described above. See, George et al. “IdiopathicThrombocytopenic Purpura: A Practice Guideline Developed by ExplicitMethods for The American Society of Hematology”, Blood 88:3-40 (1996),expressly incorporated herein by reference.

In other embodiments, antibody agonists of the invention are be used totreat, inhibit or prevent immunodeficiencies, and/or disorders, orconditions associated with immunodeficiencies. Such immunodeficienciesinclude, but are not limited to, severe combined immunodeficiency(SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADAdeficiency), X-linked agammaglobulinemia (XLA), Bruton's disease,congenital agammaglobulinemia, X-linked infantile agammaglobulinemia,acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onsetagammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,transient hypogammaglobulinemia of infancy, unspecifiedhypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell lymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, severe congenitalleukopenia, thymic alymphoplasia-aplasia or dysplasia withimmunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linkedlymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency.

In another specific embodiment, antibodies of the invention are used totreat, inhibit, prognose, diagnose or prevent CVID, or a subgroup ofindividuals having CVID.

In another specific embodiment, antibody agonists of the invention areused as an adjuvant to stimulate B cell proliferation, immunoglobulinproduction, and/or to enhance B cell survival.

The treatment and/or prevention of diseases, disorders, or conditionsassociated with aberrant expression and/or activity of a polypeptide ofthe invention includes, but is not limited to, alleviating symptomsassociated with those diseases, disorders or conditions. The antibodiesof the invention may also be used to target and kill cells expressingTR21 on their surface (e.g., B cells) and/or cells having TR21 bound totheir surface. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, or 10⁻⁸ M. Even more preferredbinding affinities include those with a dissociation constant or Kd lessthan 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹²M,10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

Gene Therapy using TR21 Antibodies

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both heavy and light chains or fragments thereof, of theantibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitate delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291-302(1994), which describes the use of a retroviral vector to deliver themdrl gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, such as, for example, anantibody of the invention. In a preferred aspect, the compound issubstantially purified (e.g., substantially free from substances thatlimit its effect or produce undesired side-effects). The subject ispreferably an animal, including but not limited to animals such as cows,pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal,and most preferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not adsorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)). Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopoeiaor other generally recognized pharmacopoeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E.W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the aberrant expression and/or activity of apolypeptide of the invention. The invention provides for the detectionof aberrant expression of a polypeptide of interest, comprising (a)assaying the expression of the polypeptide of interest in cells or bodyfluid of an individual using one or more antibodies specific to thepolypeptide interest and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed polypeptide gene expression level compared to the standardexpression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as radioisotopes, such as iodine (¹³¹I,¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium(^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (₁₃₃Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru;luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide ofinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(SIGMA™, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Diagnosis of Immune System-Related Disorders

TR21 is preferentially expressed in mature B lymphocytes. For a numberof immune system-related disorders, substantially altered (increased ordecreased) levels of TR21 gene expression may be detected in immunesystem tissue or other cells or bodily fluids (e.g., sera, plasma,urine, synovial fluid or spinal fluid) taken from an individual havingsuch a disorder, relative to a “standard” TR21 gene expression level,that is, the TR21 expression level in immune system tissues or bodilyfluids from an individual not having the immune system disorder. Thus,the invention provides a diagnostic method useful during diagnosis of animmune system disorder, which involves measuring the expression level ofthe gene encoding the TR21 polypeptide in immune system tissue or othercells or body fluid from an individual and comparing the measured geneexpression level with a standard TR21 gene expression level, whereby anincrease or decrease in the gene expression level compared to thestandard is indicative of an immune system disorder or abnormalactivation, proliferation, differentiation, and/or death.

In particular, it is believed that certain tissues in mammals withcancer of cells or tissue of the immune system express significantlyenhanced or reduced levels of the TR21 polypeptide and mRNA encoding theTR21 polypeptide when compared to a corresponding “standard” level.Further, it is believed that enhanced or depressed levels of the TR21polypeptide can be detected in certain body fluids (e.g., sera, plasma,urine, and spinal fluid) or cells or tissue from mammals with such acancer when compared to sera from mammals of the same species not havingthe cancer.

For example, as disclosed herein, TR21 is highly expressed primarily incells of B cell lineage. Accordingly, polynucleotides of the invention(e.g., polynucleotide sequences complementary to all or a portion ofTR21 mRNA) and antibodies (and antibody fragments) directed against thepolypeptides of the invention may be used to quantitate or qualitateconcentrations of cells of B cell lineage (e.g., B cell leukemia andlymphoma cells) expressing TR21 on their cell surfaces. These antibodiesadditionally have diagnostic applications in detecting abnormalities inthe level of TR21 gene expression, or abnormalities in the structureand/or temporal, tissue, cellular, or subcellular location of TR21.These diagnostic assays may be performed in vivo or in vitro, such as,for example, on blood samples, biopsy tissue or autopsy tissue.

Additionally, as disclosed herein, TR21 ligand (e.g., Neutrokine-alpha)is expressed primarily on cells of monocytic lineage. Accordingly, TR21polypeptides of the invention (including labeled TR21 polypeptides andTR21 fusion proteins), and anti-TR21 antibodies (including anti-TR21antibody fragments) against the polypeptides of the invention may beused to quantitate or qualitate concentrations of cells of monocyticlineage (e.g., monocyte cell lineage related leukemias or lymphomas)expressing Neutrokine-alpha on their cell surfaces. These TR21polypeptides and antibodies additionally have diagnostic applications indetecting abnormalities in the level of Neutrokine-alpha geneexpression, or abnormalities in the structure and/or temporal, tissue,cellular, or subcellular location of Neutrokine-alpha, and/or diagnosingactivity/defects in signaling pathways associated with TR21. Thesediagnostic assays may be performed in vivo or in vitro, such as, forexample, on blood samples or biopsy tissue using techniques describedherein or otherwise known in the art.

In one embodiment, TR21 polynucleotides or polypeptides or TR21 agonists(e.g., anti-TR21 antibodies) or antagonists (e.g., anti-TR21 antibodies)of the invention are used to treat, diagnose, or prognose an individualhaving an autoimmune disease or disorder.

Autoimmune diseases or disorders that may be treated, diagnosed, orprognosed using TR21 polynucleotides or polypeptides (e.g., TR21extracellular domain-Fc fusion polypeptides) or TR21 agonists (e.g.,anti-TR21 antibodies) or antagonists (e.g., anti-TR21 antibodies) of theinvention include, but are not limited to, one or more of the following:autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia,idiopathic thrombocytopenia purpura, autoimmune neutropenia,autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,dermatitis, gluten sensitive enteropathy, allergic encephalomyelitis,myocarditis, relapsing polychondritis, rheumatic heart disease,glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis,Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g.,Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome,Autoimmune Pulmonary Inflammation, myocarditis, IgA glomerulonephritis,dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome,insulin dependent diabetes mellitis, and autoimmune inflammatory eye,autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis,systemic lupus erhythematosus, discoid lupus, Goodpasture's syndrome,Pemphigus, Receptor autoimmunities such as, for example, (a) Graves'Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmunehemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoidarthritis, scleroderma with anti-collagen antibodies, mixed connectivetissue disease, polymyositis/dermatomyositis, pernicious anemia,idiopathic Addison's disease, infertility, glomerulonephritis such asprimary glomerulonephritis and IgA nephropathy, bullous pemphigoid,Sjogren's syndrome, diabetes mellitus, and adrenergic drug resistance(including adrenergic drug resistance with asthma or cystic fibrosis),chronic active hepatitis, primary biliary cirrhosis, other endocrinegland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome,urticaria, atopic dermatitis, asthma, inflammatory myopathies, and otherinflammatory, granulamatous, degenerative, and atrophic disorders.

According to this embodiment, an individual having an autoimmune diseaseor disorder may express aberrantly high levels of TR21 ligand (e.g.,Neutrokine-alpha) and/or TR21 when compared to an individual not havingan autoimmune disease or disorder. Any means described herein orotherwise known in the art may be applied to detect TR21 polynucleotidesor polypeptides of the invention (e.g., FACS analysis or ELISA detectionof TR21 polypeptides of the invention and hybridization or PCR detectionof TR21 polynucleotides of the invention) and to determine theexpression profile of, for example, TR21, polynucleotides and/orpolypeptides of the invention, in a biological sample.

A biological sample of persons afflicted with an autoimmune disease ordisorder may be characterized by high levels of expression of TR21 whencompared to that observed in individuals not having an autoimmunedisease or disorder. Thus, TR21 polynucleotides and/or polypeptides(e.g., anti-TR21 antibodies and TR21-extracellular domain-Fc fusionpolypeptides) of the invention and/or agonists or antagonists thereof,may be used according to the methods of the invention in the diagnosisand/or prognosis of an autoimmune disease or disorder. For example, abiological sample obtained from a person suspected of being afflictedwith an autoimmune disease or disorder (“the subject”) may be analyzedfor the relative expression level(s) of TR21 polynucleotides and/orpolypeptides of the invention. The expression level(s) of one or more ofthe TR21 molecules of the invention is (are) then compared to theexpression level(s) of the same molecules of the invention as expressedin a person known not to be afflicted with an autoimmune disease ordisorder. According to this example, a significant difference inexpression level(s) of TR21, polynucleotides and/or polypeptides of theinvention, and/or agonists and/or antagonists thereof, between samplesobtained from the subject and the control suggests that the subject isafflicted with an autoimmune disease or disorder.

In another embodiment, TR21 polynucleotides or polypeptides or TR21agonists (such as, for example, anti-TR21 antibodies) or TR21antagonists (such as, for example, anti-TR21 antibodies) of theinvention are used to treat, diagnose, or prognose an individual havingsystemic lupus erythematosus or a subset of this disease. According tothis embodiment, an individual having systemic lupus erythematosus or asubset of individuals having systemic lupus erythematosus may expressaberrantly high levels of TR21 when compared to an individual not havingsystemic lupus erythematosus or this subset of systemic lupus. TR21polynucleotides or polypeptides of the invention (e.g., FACS analysis orELISA detection of TR21 polypeptides of the invention and hybridizationor PCR detection of TR21 polynucleotides of the invention) may be usedto determine the expression profile of TR21, polynucleotides and/orpolypeptides of the invention in a biological sample.

A biological sample of persons afflicted with systemic lupuserythematosus may be characterized by high levels of expression of TR21when compared to that observed in individuals not having systemic lupuserythematosus. Thus, TR21 polynucleotides and/or polypeptides of theinvention and/or agonists or antagonists thereof, may be used accordingto the methods of the invention in the diagnosis and/or prognosis ofsystemic lupus erythematosus or a subset of systemic lupuserythematosus. For example, a biological sample obtained from a personsuspected of being afflicted with systemic lupus erythematosus (“thesubject”) may be analyzed for the relative expression level(s) of TR21polynucleotides and/or polypeptides of the invention. The expressionlevel(s) of one or more of these molecules of the invention is (are)then compared to the expression level(s) of the same molecules of theinvention as expressed in a person known not to be afflicted withsystemic lupus erythematosus. According to this example, a significantdifference in expression level(s) of TR21, polynucleotides and/orpolypeptides of the invention, and/or agonists (e.g., agonisticantibodies) and/or antagonists thereof, between samples obtained fromthe subject and the control suggests that the subject is afflicted withsystemic lupus erythematosus or a subset thereof.

Furthermore, there may be a direct correlation between the severity ofsystemic lupus erythematosus, or a subset of this disease, and theconcentration of TR21 polynucleotides (RNA) and/or polypeptides of theinvention. Thus, TR21 polynucleotides (RNA) and/or polypeptides and/oragonists or antagonists of the invention, may be used according to themethods of the invention in prognosis of the severity of systemic lupuserythematosus or a subset of systemic lupus erythematosus. For example,a biological sample obtained from a person suspected of being afflictedwith systemic lupus erythematosus (“the subject”) may be analyzed forthe relative expression level(s) of TR21 polynucleotides and/orpolypeptides of the invention. The expression level(s) of one or more ofthese molecules of the invention is (are) then compared to theexpression level(s) of the same molecules of the invention as expressedin a panel of persons known to represent a range in severities of thisdisease. According to this example, the match of expression level with acharacterized member of the panel indicates the severity of the disease.

In another embodiment, TR21 polynucleotides or polypeptide (e.g.,anti-TR21 antibodies and TR21 extracellular domain-Fc fusionpolypeptides) or TR21 agonists (such as, for example, anti-TR21antibodies) or TR21 antagonists (such as, for example, anti-TR21antibodies) of the invention are used to treat, diagnose, or prognose anindividual having rheumatoid arthritis or a subset of this disease.According to this embodiment, an individual having rheumatoid arthritisor a subset of individuals having rheumatoid arthritis may expressaberrantly high levels of TR21 when compared to an individual not havingrheumatoid arthritis or this subset of rheumatoid arthritis. Any meansdescribed herein or otherwise known in the art may be applied to detectTR21 polynucleotides or polypeptides of the invention (e.g., FACSanalysis or ELISA detection of TR21 polypeptides of the invention andhybridization or PCR detection of TR21 polynucleotides of the invention)and to determine the expression profile of TR21 polynucleotides and/orpolypeptides of the invention) in a biological sample.

A biological sample of persons afflicted with rheumatoid arthritis maybe characterized by high levels of expression of TR21 when compared tothat observed in individuals not having rheumatoid arthritis. Thus, TR21polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof, may be used according to the methods of theinvention in the diagnosis and/or prognosis of rheumatoid arthritis or asubset of rheumatoid arthritis. For example, a biological sampleobtained from a person suspected of being afflicted with rheumatoidarthritis (“the subject”) may be analyzed for the relative expressionlevel(s) of TR21 polynucleotides and/or polypeptides of the invention.The expression level(s) of one or more of these molecules of theinvention is (are) then compared to the expression level(s) of the samemolecules of the invention as expressed in a person known not to beafflicted with rheumatoid arthritis. According to this example, asignificant difference in expression level(s) of TR21, polynucleotidesand/or polypeptides of the invention, and/or agonists and/or antagoniststhereof, between samples obtained from the subject and the controlsuggests that the subject is afflicted with rheumatoid arthritis or asubset thereof.

In another embodiment, TR21 polynucleotides or polypeptides or TR21agonists (e.g., anti-TR21 antibodies) or antagonists (e.g., anti-TR21antibodies) of the invention are used to treat, prevent, diagnose, orprognose an individual having an immunodeficiency.

Immunodeficiencies that may be treated, prevented, diagnosed, and/orprognosed with the TR21 polynucleotides or polypeptides or TR21 agonists(e.g., anti-TR21 antibodies) or antagonists (e.g., anti-TR21 antibodies)of the invention, include, but are not limited to one or moreimmunodeficiencies selected from: severe combined immunodeficiency(SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADAdeficiency), X-linked agammaglobulinemia (XLA), Bruton's disease,congenital agammaglobulinemia, X-linked infantile agammaglobulinemia,acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onsetagammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,transient hypogammaglobulinemia of infancy, unspecifiedhypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVID) (acquired), chronic granulomatous disease,Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency with hyperIgM, non X-linked immunodeficiency with hyper IgM, selective IgAdeficiency, IgG subclass deficiency (with or without IgA deficiency),antibody deficiency with normal or elevated Igs, immunodeficiency withthymoma, Ig heavy chain deletions, kappa chain deficiency, B celllymphoproliferative disorder (BLPD), selective IgM immunodeficiency,recessive agammaglobulinemia (Swiss type), reticular dysgenesis,neonatal neutropenia, severe congenital leukopenia, thymicalymphoplasia-aplasia or dysplasia with immunodeficiency,ataxia-telangiectasia, short limbed dwarfism, X-linkedlymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency.

According to this embodiment, an individual having an immunodeficiencymay express aberrantly low levels of TR21 when compared to an individualnot having an immunodeficiency. Any means described herein or otherwiseknown in the art may be applied to detect TR21 polynucleotides orpolypeptides of the invention (e.g., FACS analysis or ELISA detection ofTR21 polypeptides of the invention and hybridization or PCR detection ofTR21 polynucleotides of the invention) and to determine the expressionprofile of TR21 polynucleotides and/or polypeptides of the invention ina biological sample.

A biological sample of a person afflicted with an immunodeficiency maybe characterized by low levels of expression of TR21 when compared tothat observed in individuals not having an immunodeficiency. Thus, TR21polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof, may be used according to the methods of theinvention in the diagnosis and/or prognosis of an immunodeficiency. Forexample, a biological sample obtained from a person suspected of beingafflicted with an immunodeficiency (“the subject”) may be analyzed forthe relative expression level(s) of TR21 polynucleotides and/orpolypeptides of the invention. The expression level(s) of one or more ofthese molecules of the invention is (are) then compared to theexpression level(s) of the same molecules of the invention as expressedin a person known not to be afflicted with an immunodeficiency. Asignificant difference in expression level(s) of TR21, polynucleotidesand/or polypeptides of the invention, and/or agonists and/or antagoniststhereof, between samples obtained from the subject and the controlsuggests that the subject is afflicted with an immunodeficiency.

In another embodiment, TR21 polynucleotides or polypeptides (e.g., TR21extracellular domain-Fc fusion polypeptides) or TR21 agonists (e.g.,anti-TR21 antibodies) or antagonists (e.g., anti-TR21 antibodies) of theinvention are used to treat, diagnose and/or prognose an individualhaving common variable immunodeficiency disease (“CVID”; also known as“acquired agammaglobulinemia” and “acquired hypogammaglobulinemia”) or asubset of this disease. According to this embodiment, an individualhaving CVID or a subset of individuals having CVID expresses aberrantlevels of TR21 and/or Neutrokine-alpha or Neutrokine-alpha SV on their Bcells and/or monocytes, when compared to individuals not having CVID.Any means described herein or otherwise known in the art may be appliedto detect TR21 polynucleotides or polypeptides of the invention (e.g.,FACS analysis or ELISA detection of TR21 polypeptides of the inventionand hybridization or PCR detection of TR21 polynucleotides of theinvention) and to determine differentially the expression profile ofTR21 polynucleotides or polypeptides of the invention in a samplecontaining at least monocyte cells or some component thereof (e.g., RNA)as compared to a sample containing at least B cells or a componentthereof (e.g., RNA). In the instance where a sample containing at leastmonocyte cells or some component thereof (e.g., RNA) is determined toreflect TR21 ligand (e.g., Neutrokine-alpha) polynucleotide orpolypeptide expression and a sample containing at least B cells or acomponent thereof (e.g., RNA) is determined to reflect less than normallevels of TR21 polynucleotide or polypeptide expression, the samples maybe correlated with the occurrence of CVID (i.e., “acquiredagammaglobulinemia” or “acquired hypogammaglobulinemia”).

A subset of persons afflicted with CVID may be characterized by highlevels of expression of Neutrokine-alpha, Neutrokine-alpha SV, and/orTR21 polypeptides in peripheral or circulating B cells when compared tothat observed in individuals not having CVID. In contrast, persons whoare not afflicted with CVID are typically characterized by low levels ofNeutrokine-alpha expression and high levels of TR21 expression inperipheral or circulating B cells. Thus, TR21 polypeptides,polynucleotides and/or polypeptides of the invention, and/or agonists orantagonists thereof, may be used according to the methods of theinvention in the differential diagnosis of this subset of CVID. Forexample, a sample of peripheral B cells obtained from a person suspectedof being afflicted with CVID (“the subject”) may be analyzed for therelative expression level(s) of Neutrokine-alpha, Neutrokine-alpha SV,and/or TR21 polynucleotides and/or polypeptides of the invention. Theexpression level(s) of one or more of these molecules of the inventionis (are) then compared to the expression level(s) of the same moleculesof the invention as expressed in a person known not to be afflicted withCVID (“the control”). According to this example, a significantdifference in expression level(s) of Neutrokine-alpha, Neutrokine-alphaSV, and/or TR21 polynucleotides or polypeptides of the invention, and/oragonists and/or antagonists thereof, between samples obtained from thesubject and the control suggests that the subject is afflicted with thissubset of CVID.

Cunningham-Rundles and Bodian followed 248 CVID patients over a periodof 1-25 years and discovered that a number of associated diseases orconditions appear with increased frequency in CVID patients(Cunningham-Rundles and Bodian, J. Clin. Immunol., 92:34-48 (1999) whichis herein incorporated by reference in its entirety.) The most importantclinical events include infections, autoimmunity, inflammatorydisorders, marked by gastrointestinal and granulomatous disease, cancerand hepatitis. Most CVID patients are at increased risk of recurrentinfections particularly of the respiratory tract. The types of acute andrecurring bacterial infections exhibited in most patients includepneumonia, bronchitis and sinusitis. Children with CVID have a markedincreased risk of otitis media. Additionally, blood borne infectionsincluding sepsis, meningitis, septic arthritis, and osteomyelitis areseen with increased frequency in these patients.

In another specific embodiment, TR21 polynucleotides or polypeptides, oragonists or antagonists thereof (e.g., anti-TR21 antibodies) are used todiagnose, prognose, treat, or prevent conditions associated with CVID,including, but not limited to, conditions associated with acute andrecurring infections (e.g., pneumonia, bronchitis, sinusitis, otitismedia, sepsis, meningitis, septic arthritis, and osteomyelitis), chroniclung disease, autoimmunity, granulomatous disease, lymphoma, cancers(e.g., cancers of the breast, stomach, colon, mouth, prostate, lung,vagina, ovary, skin, and melanin forming cells (i.e. melanoma),inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis,and ulcerative proctitis), malabsorption, Hodgkin's disease, andWaldenstrom's macroglobulinemia.

In a specific embodiment, TR21 polynucleotides or polypeptides, oragonists or antagonists thereof (e.g., anti-TR21 antibodies) are used todiagnose, prognose, treat, or prevent a disorder characterized bydeficient serum immunoglobulin production, recurrent infections, and/orimmune system dysfunction. Moreover, TR21 polynucleotides orpolypeptides, or agonists or antagonists thereof (e.g., anti-TR21antibodies) may be used to diagnose, prognose, treat, or preventinfections of the joints, bones, skin, and/or parotid glands,blood-borne infections (e.g., sepsis, meningitis, septic arthritis,and/or osteomyelitis), autoimmune diseases (e.g., those disclosedherein), inflammatory disorders, and malignancies, and/or any disease ordisorder or condition associated with these infections, diseases,disorders and/or malignancies) including, but not limited to, CVID,other primary immune deficiencies, HIV disease, CLL, recurrentbronchitis, sinusitis, otitis media, conjunctivitis, pneumonia,hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster),and/or pneumocystis carnii.

Thus, the invention provides a diagnostic method useful during diagnosisof a immune system disorder, including cancers of this system, andimmunodeficiencies and/or autoimmune diseases which involves measuringthe expression level of the gene encoding TR21 polypeptide in immunesystem tissue or other cells or body fluid from an individual andcomparing the measured gene expression level with a standard TR21 geneexpression level, whereby an increase or decrease in the gene expressionlevel compared to the standard is indicative of an immune systemdisorder.

Where a diagnosis of a disorder in the immune system, including, but notlimited to, diagnosis of a tumor, diagnosis of an immunodeficiency,and/or diagnosis of an autoimmune disease, has already been madeaccording to conventional methods, the present invention is useful as aprognostic indicator, whereby patients exhibiting enhanced or depressedTR21 gene expression will experience a worse clinical outcome relativeto patients expressing the gene at a level nearer the standard level.

By analyzing or determining the expression level of the gene encodingthe TR21 polypeptide is intended qualitatively or quantitativelymeasuring or estimating the level of the TR21 polypeptide or the levelof the mRNA encoding the TR21 polypeptide in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing to the TR21polypeptide level or mRNA level in a second biological sample).Preferably, the TR21 polypeptide level or mRNA level in the firstbiological sample is measured or estimated and compared to a standardTR21 polypeptide level or mRNA level, the standard being taken from asecond biological sample obtained from an individual not having thedisorder or being determined by averaging levels from a population ofindividuals not having a disorder of the immune system. As will beappreciated in the art, once a standard TR21 polypeptide level or mRNAlevel is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains TR21 polypeptide or mRNA. As indicated, biologicalsamples include body fluids (such as sera, plasma, urine, synovial fluidand spinal fluid) which contain free extracellular domains of the TR21polypeptide, immune system tissue, and other tissue sources found toexpress complete or free extracellular domain of the TR21. Methods forobtaining tissue biopsies and body fluids from mammals are well known inthe art. Where the biological sample is to include mRNA, a tissue biopsyis the preferred source.

The compounds of the present invention are useful for diagnosis,prognosis, or treatment of various immune system-related disorders inmammals, preferably humans. Such disorders include, but are not limitedto tumors (e.g., B cell and monocytic cell leukemias and lymphomas) andtumor metastasis, infections by bacteria, viruses and other parasites,immunodeficiencies, inflammatory diseases, lymphadenopathy, autoimmunediseases (e.g., rheumatoid arthritis, systemic lupus erythematosus,Sjogren's syndrome, mixed connective tissue disease, and inflammatorymyopathies), and graft versus host disease.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the TR21 polypeptide are then assayed using anyappropriate method. These include Northern blot analysis, S1 nucleasemapping, the polymerase chain reaction (PCR), reverse transcription incombination with the polymerase chain reaction (RT-PCR), and reversetranscription in combination with the ligase chain reaction (RT-LCR).

Assaying TR21 polypeptide levels in a biological sample can occur usingantibody-based techniques. For example, TR21 polypeptide expression intissues can be studied with classical immunohistological methods(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-basedmethods useful for detecting TR21 polypeptide gene expression includeimmunoassays, such as the enzyme linked immunosorbent assay (ELISA) andthe radioimmunoassay (RIA). Suitable antibody assay labels are known inthe art and include enzyme labels, such as, glucose oxidase, andradioisotopes, such as iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In),and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium, (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F),¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru; luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin.

Techniques known in the art may be applied to label polypeptides(including antibodies) of the invention. Such techniques include, butare not limited to, the use of bifunctional conjugating agents (seee.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361;5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119;4,994,560; and 5,808,003; the contents of each of which are herebyincorporated by reference in its entirety).

The tissue or cell type to be analyzed will generally include thosewhich are known, or suspected, to express the TR21 (such as, forexample, cells of B cell lineage and the spleen). The protein isolationmethods employed herein may, for example, be such as those described inHarlow and Lane (Harlow, E. and Lane, D., 1988, “Antibodies: ALaboratory Manual”, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.), which is incorporated herein by reference in itsentirety. The isolated cells can be derived from cell culture or from apatient. The analysis of cells taken from culture may be a necessarystep in the assessment of cells that could be used as part of acell-based gene therapy technique or, alternatively, to test the effectof compounds on the expression of the TR21 gene.

For example, antibodies, or fragments of antibodies, such as thosedescribed herein, may be used to quantitatively or qualitatively detectthe presence of TR21 gene products or conserved variants or peptidefragments thereof. This can be accomplished, for example, byimmunofluorescence techniques employing a fluorescently labeled antibodycoupled with light microscopic, flow cytometric, or fluorimetricdetection.

The antibodies (or fragments thereof) or TR21 polynucleotides orpolypeptides, may additionally be employed histologically, as inimmunofluorescence, immunoelectron microscopy or non-immunologicalassays, for in situ detection of TR21 gene products or conservedvariants or peptide fragments thereof, or for Neutrokine-alpha and/orNeutrokine-alpha SV binding to TR21. In situ detection may beaccomplished by removing a histological specimen from a patient, andapplying thereto a labeled antibody or TR21 polypeptide of the presentinvention. The antibody (or fragment) or TR21 polypeptide is preferablyapplied by overlaying the labeled antibody (or fragment) onto abiological sample. Through the use of such a procedure, it is possibleto determine not only the presence of the TR21 gene product, orconserved variants or peptide fragments, or TR21 polypeptide binding,but also its distribution in the examined tissue. Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

Immunoassays and non-immunoassays for TR21 gene products or conservedvariants or peptide fragments thereof will typically comprise incubatinga sample, such as a biological fluid, a tissue extract, freshlyharvested cells, or lysates of cells which have been incubated in cellculture, in the presence of a detectably labeled antibody capable ofidentifying TR21 gene products or conserved variants or peptidefragments thereof, and detecting the bound antibody by any of a numberof techniques well-known in the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled an anti-TR21 antibodyor detectable polypeptide. The solid phase support may then be washedwith the buffer a second time to remove unbound antibody or polypeptide.Optionally the antibody is subsequently labeled. The amount of boundlabel on solid support may then be detected by conventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-TR21 antibody or TR21polypeptide may be determined according to well-known methods. Thoseskilled in the art will be able to determine operative and optimal assayconditions for each determination by employing routine experimentation.

In addition to assaying TR21 polypeptide levels or polynucleotide levelsin a biological sample obtained from an individual, TR21 polypeptides orpolynucleotides can also be detected in vivo by imaging. For example, inone embodiment of the invention, TR21 polypeptide and/or anti-TR21antibody is used to image B cell lymphomas. In another embodiment, TR21polypeptides and/or anti-TR21 antibodies and/or TR21 polynucleotides ofthe invention (e.g., polynucleotides complementary to all or a portionof TR21 mRNA) is used to image lymphomas (e.g., monocyte and B celllymphomas).

Antibody labels or markers for in vivo imaging of TR21 polypeptideinclude those detectable by X-radiography, NMR, MRI, CAT-scans or ESR.For X-radiography, suitable labels include radioisotopes such as bariumor cesium, which emit detectable radiation but are not overtly harmfulto the subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma. Where in vivo imaging is used to detect enhanced levels ofTR21 polypeptide for diagnosis in humans, it may be preferable to usehuman antibodies or “humanized” chimeric monoclonal antibodies. Suchantibodies can be produced using techniques described herein orotherwise known in the art. For example methods for producing chimericantibodies are known in the art. See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature314:268 (1985).

Additionally, any TR21 polypeptide whose presence can be detected can beadministered. For example, TR21 polypeptides labeled with a radio-opaqueor other appropriate compound can be administered and visualized invivo, as discussed, above for labeled antibodies. Further such TR21polypeptides can be utilized for in vitro diagnostic procedures.

A TR21 polypeptide-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc, (¹³¹I, ¹²⁵I, ¹²³I,¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^(115m) _(In,)^(113m)In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium(²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo),xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb,¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for immune systemdisorder. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ^(99m)Tc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain TR21 protein. In vivotumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

With respect to antibodies, one of the ways in which the anti-TR21antibody can be detectably labeled is by linking the same to an enzymeand using the linked product in an enzyme immunoassay (EIA) (Voller, A.,“The Enzyme Linked Immunosorbent Assay (ELISA)”, 1978, DiagnosticHorizons 2:1-7, Microbiological Associates Quarterly Publication,Walkersville, Md.); Voller et al., J. Clin. Pathol. 31:507-520 (1978);Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980,Enzyme Immunoassay, CRC Press, Boca Raton, Fla.; Ishikawa, E. et al.,(eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme whichis bound to the antibody will react with an appropriate substrate,preferably a chromogenic substrate, in such a manner as to produce achemical moiety which can be detected, for example, byspectrophotometric, fluorimetric or by visual means. Enzymes which canbe used to detectably label the antibody include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate,dehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. Additionally, the detection canbe accomplished by colorimetric methods which employ a chromogenicsubstrate for the enzyme. Detection may also be accomplished by visualcomparison of the extent of enzymatic reaction of a substrate incomparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect TR21 through the use of aradioimmunoassay (RIA) (see, for example, Weintraub, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March, 1986, which is incorporated byreference herein). The radioactive isotope can be detected by meansincluding, but not limited to, a gamma counter, a scintillation counter,or autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wavelength, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling include, but are not limited to, luciferin, luciferase andaequorin.

Treatment of Immune System-Related Disorders

As noted above, TR21 polynucleotides and polypeptides (e.g., TR21extracellular domain-Fc fusion proteins), and anti-TR21 antibodies, areuseful for diagnosis of conditions involving abnormally high or lowexpression of TR21 activities. For example, given the cells and tissueswhere TR21 is expressed as well as the activities modulated by TR21, itis readily apparent that a substantially altered (increased ordecreased) level of expression of TR21 in an individual compared to thestandard or “normal” level may produce pathological conditions relatedto the bodily system(s) in which TR21 is expressed and/or is active.

It will also be appreciated by one of ordinary skill that, since theTR21 polypeptides of the invention are members of the TNFR family, theextracellular domains of the respective proteins may be released insoluble form from the cells which express TR21 by proteolytic cleavageand therefore, when TR21 polypeptide (particularly a soluble form of therespective extracellular domains) is added from an exogenous source tocells, tissues or the body of an individual, the polypeptide may inhibitthe modulating activities of its ligand (e.g., Neutrokine-alpha) on anyof its target cells of that individual. Also, cells expressing this typeIII transmembrane protein may be added to cells, tissues or the body ofan individual whereby the added cells will bind to cells expressing TR21ligands (e.g., Neutrokine-alpha) whereby the cells expressing the TR21ligand (e.g., Neutrokine-alpha) can cause actions (e.g., proliferationor cytotoxicity) on the ligand-bearing target cells.

The present invention is further directed to TR21 based therapies whichinvolve administering TR21 based therapeutic compounds of the inventionto an animal, preferably a mammal, and most preferably a human, patientfor treating one or more of the diseases, disorders, or conditionsdisclosed herein. Therapeutic compounds of the invention include, butare not limited to, TR21 polypeptides (including fragments and variantsof TR21 polypeptides), polynucleotides encoding these polypeptides,antibodies that bind these polypeptides, and agonists and/or antagonistsof these polypeptides, polynucleotides and antibodies. The TR21polypeptides, polynucleotides, and antibodies of the invention can beused to treat, ameliorate or prevent diseases, disorders or conditionsassociated with aberrant expression and/or activity of Neutrokine-alpha,Neutrokine-alpha SV (See, e.g., U.S. Pat. No. 5,969,102; and von Bulowet al., Science 278:138-141 (1997)), and/or TR21, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of Neutrokine-alpha, Neutrokine-alpha SV, and/or TR21,includes, but is not limited to, alleviating symptoms associated withthose diseases, disorders or conditions. TR21 compositions of theinvention may be provided in pharmaceutically acceptable compositions asknown in the art or as described herein.

The TR21 polypeptides, polynucleotides, and antibodies of the inventionthat function as agonists or antagonists of Neutrokine alpha,Neutrokine-alpha SV and/or heteromultimeric polypeptide complexescomprising one or more copies of Neutrokine-alpha and/orNeutrokine-alpha SV, preferably of signal transduction induced byNeutrokine alpha, Neutrokine-alpha SV and/or heteromultimericpolypeptide complexes comprising one or more copies of Neutrokine-alphaand/or Neutrokine-alpha SV, can be administered to an animal to treat,prevent or ameliorate a disease or disorder associated with aberrantexpression of Neutrokine alpha, Neutrokine-alpha SV and/orheteromultimeric polypeptide complexes comprising one or more copies ofNeutrokine-alpha and/or Neutrokine-alpha SV, lack of function ofNeutrokine alpha, Neutrokine-alpha SV and/or heteromultimericpolypeptide complexes comprising one or more copies of Neutrokine-alphaand/or Neutrokine-alpha SV, aberrant TR21 expression, aberrant TACIand/or BCMA expression, lack of TR21 function and/or lack of TACI and/orBCMA function. For example, TR21 polypeptides of the invention whichdisrupt the interaction between TR21 and one or more of its ligands maybe administered to an animal to treat, prevent or ameliorate a diseaseor disorder associated with aberrant expression of Neutrokine alpha,Neutrokine-alpha SV and/or heteromultimeric polypeptide complexescomprising one or more copies of Neutrokine-alpha and/orNeutrokine-alpha SV, excessive function of Neutrokine alpha,Neutrokine-alpha SV and/or heteromultimeric polypeptide complexescomprising one or more copies of Neutrokine-alpha and/orNeutrokine-alpha SV, aberrant TR21 expression, aberrant TACI and/or BCMAexpression, excessive TR21 function or excessive TACI and/or BCMAfunction.

In a preferred embodiment, TR21 polypeptides of the invention neutralizeactivity of Neutrokine alpha, Neutrokine-alpha SV and/orheteromultimeric polypeptide complexes comprising one or more copies ofNeutrokine-alpha and/or Neutrokine-alpha SV. In another preferredembodiment, TR21 polypeptides of the invention inhibit B cellproliferation. In another preferred embodiment, TR21 polypeptides of theinvention inhibit immunoglobulin production by B cells.

In a preferred embodiment, TR21 polypeptides of the invention (includingTR21 fragments and variants, and anti-TR21 antibodies) inhibit or reducebinding of the soluble form of Neutrokine alpha, Neutrokine-alpha SVand/or heteromultimeric polypeptide complexes comprising one or morecopies of Neutrokine-alpha and/or Neutrokine-alpha SV to a Neutrokinealpha receptor (e.g., TR21 and/or TACI and/or BCMA). In anotherpreferred embodiment TR21 polypeptides of the invention (including TR21fragments and variants, and anti-TR21 antibodies) inhibit or reduce Bcell proliferation induced by the soluble form of Neutrokine alpha,Neutrokine-alpha SV and/or heteromultimeric polypeptide complexescomprising one or more copies of Neutrokine-alpha and/orNeutrokine-alpha SV. In another preferred embodiment TR21 polypeptidesof the invention (including TR21 fragments and variants, and anti-TR21antibodies) inhibit or reduce immunoglobulin production induced by thesoluble form of Neutrokine alpha, Neutrokine-alpha SV and/orheteromultimeric polypeptide complexes comprising one or more copies ofNeutrokine-alpha and/or Neutrokine-alpha SV. In another preferredembodiment TR21 polypeptides of the invention (including TR21 fragmentsand variants, and anti-TR21 antibodies) inhibit or reduce immunoglobulinproduction in response to T cell dependent immunogens. In anotherpreferred embodiment TR21 polypeptides of the invention (including TR21fragments and variants, and anti-TR21 antibodies) inhibit or reduceimmunoglobulin production in response to T cell independent immunogens.

In another preferred embodiment TR21 polypeptides of the invention(including TR21 fragments and variants, and anti-TR21 antibodies)promote or enhance B cell proliferation induced by the soluble form ofNeutrokine alpha, Neutrokine-alpha SV and/or heteromultimericpolypeptide complexes comprising one or more copies of Neutrokine-alphaand/or Neutrokine-alpha SV. In another preferred embodiment TR21polypeptides of the invention (including TR21 fragments and variants,and anti-TR21 antibodies) increase or enhance immunoglobulin productioninduced by the soluble form of Neutrokine alpha, Neutrokine-alpha SVand/or heteromultimeric polypeptide complexes comprising one or morecopies of Neutrokine-alpha and/or Neutrokine-alpha SV. In anotherpreferred embodiment TR21 polypeptides of the invention (including TR21fragments and variants, and anti-TR21 antibodies) increase or enhanceimmunoglobulin production in response to T cell dependent immunogens. Inanother preferred embodiment TR21 polypeptides of the invention(including TR21 fragments and variants, and anti-TR21 antibodies)increase or enhance immunoglobulin production in response to T cellindependent immunogens.

In one embodiment, the invention provides a method of deliveringradiolabeled TR21 (including TR21 fragments and variants, and anti-TR21antibodies) polypeptide conjugates of the invention to targeted cells,such as, for example, monocytic cells expressing membrane-bound forms ofNeutrokine alpha, Neutrokine-alpha SV and/or heteromultimericpolypeptide complexes comprising one or more copies of Neutrokine-alphaand/or Neutrokine-alpha SV.

In one embodiment, the invention provides methods and compositions forinhibiting or reducing immunoglobulin production (e.g. IgM, IgG, and/orIgA production), comprising, or alternatively consisting of, contactingan effective amount of TR21 polypeptides of the invention (includingTR21 fragments and variants, and anti-TR21 antibodies) with Neutrokinealpha, Neutrokine-alpha SV and/or heteromultimeric polypeptide complexescomprising one or more copies of Neutrokine-alpha and/orNeutrokine-alpha SV, wherein the effective amount of TR21 polypeptideinhibits or reduces immunoglobulin production mediated by Neutrokinealpha, Neutrokine-alpha SV and/or heteromultimeric polypeptide complexescomprising one or more copies of Neutrokine-alpha and/orNeutrokine-alpha SV. In another embodiment, the invention providesmethods and compositions for inhibiting or reducing immunoglobulinproduction (e.g. IgM, IgG, and/or IgA production), comprising, oralternatively consisting of, administering to an animal in which suchinhibition or reduction is desired, TR21 polypeptides of the invention(including TR21 fragments and variants, and anti-TR21 antibodies) in anamount effective to inhibit or reduce immunoglobulin production.

In another embodiment, the invention provides methods and compositionsfor stimulating immunoglobulin production (e.g. IgM, IgG, and/or IgAproduction), comprising, or alternatively consisting of, contacting aneffective amount of TR21 polypeptides of the invention (including TR21fragments and variants, and anti-TR21 antibodies) with Neutrokine alpha,Neutrokine-alpha SV and/or heteromultimeric polypeptide complexescomprising one or more copies of Neutrokine-alpha and/orNeutrokine-alpha SV, wherein the effective amount of the TR21polypeptide stimulates immunoglobulin production mediated by Neutrokinealpha, Neutrokine-alpha SV and/or heteromultimeric polypeptide complexescomprising one or more copies of Neutrokine-alpha and/orNeutrokine-alpha SV. In another embodiment, the invention providesmethods and compositions for stimulating immunoglobulin production (e.g.IgM, IgG, and/or IgA production) comprising, or alternatively consistingof, administering to an animal in which such stimulation is desired, aTR21 polypeptide of the invention (including TR21 fragments andvariants, and anti-TR21 antibodies) in an amount effective to stimulateimmunoglobulin production. Determination of immunoglobulin levels aremost often performed by comparing the level of immunoglobulin in asample to a standard containing a known amount of immunoglobulin usingELISA assays. Determination of immunoglobulin levels in a given samplecan readily be determined using ELISA or other method known in the art.

Investigation of Neutrokine-alpha induced signaling in human tonsillar Bcells co-stimulated with Staphylococcus aureus Cowan consistentlyrevealed that mRNA for ERK-1 and PLK were upregulated by treatment withNeutrokine-alpha combined with SAC (data not shown). Polo like kinases(PLK) belong to a sub family of serine/threonine kinases related to theSaccharomyces cerevisiae cell cycle protein CDC5. The expression of PLKis induced during G2 and S phase of the cell cycle. PLK is reported toplay a role in cell proliferation (See e.g., Lee et al., Proc. Natl.Acad. Sci. 95:9301-9306). The role of extracellular-signal relatedkinases 1 and 2 (ERK1 and 2) in cell survival and proliferation inresponse to stimulation by growth factors and other agonists has beenextensively studied. The induced expression of PLK and ERK-1 isconsistent with the survival and proliferative effects ofNeutrokine-alpha on B cells.

Additionally, in some samples of human tonsillar B cells stimulated withNeutrokine-alpha and SAC, mRNA for CD25 (IL-2R alpha) was upregulated.Nuclear extracts from human tonsillar B cells treated withNeutrokine-alpha and from IM-9 cells treated with Neutrokine-alpha wereable to shift probes from the CD25 promoter region containing sites forNF-kappaB, SRF, ELF-1 and HMGI/Y in an electromobility shift assay.ELF-1 for example, is a transcription factor that is part of the ETSfamily of proteins and whose expression appears to be restricted to Tand B cells. Binding sites for ELF-1 have been described in thepromoters of a number of proteins that are important in the regulationof the immune response.

Thus Neutrokine-alpha induced signaling has been shown to be consistentwith the activation of cellular activation and cellular proliferationpathways as well as with cellular signaling pathways that regulate Bcell lifespan. Further, Neutrokine-alpha treatment of B cells inducescellular proliferation and immunoglobulin secretion, a characteristic ofactivation of B cells (Moore et al., Science 285:260-263, 1999). TR21polypeptides complexed with Neutrokine-alpha may inhibit, stimulate, ornot significantly alter these Neutrokine-alpha mediated activities.

In one embodiment, the invention provides methods and compositions forinhibiting or reducing B cell proliferation, comprising, oralternatively consisting of, contacting an effective amount of TR21polypeptides of the invention (including TR21 fragments and variants,and anti-TR21 antibodies) with Neutrokine-alpha, wherein the effectiveamount of TR21 polypeptide inhibits or reduces Neutrokine-alpha mediatedB cell proliferation.

In another embodiment, the invention provides methods and compositionsfor inhibiting or reducing B cell proliferation, comprising, oralternatively consisting of, contacting an effective amount of TR21polypeptides of the invention (including TR21 fragments and variants,and anti-TR21 antibodies) with Neutrokine-alpha SV, wherein theeffective amount of TR21 polypeptide inhibits or reducesNeutrokine-alpha SV mediated B cell proliferation.

In another embodiment, the invention provides methods and compositionsfor inhibiting or reducing B cell proliferation, comprising, oralternatively consisting of, contacting an effective amount of TR21polypeptides of the invention (including TR21 fragments and variants,and anti-TR21 antibodies) with one or more heteromultimeric polypeptidecomplexes comprising one or more Neutrokine-alpha and/orNeutrokine-alpha SV polypeptides, wherein the effective amount of TR21polypeptide inhibits or reduces B cell proliferation mediated by saidhetermiultimeric polypeptide complexes.

In another embodiment, the invention provides methods and compositionsfor inhibiting or reducing B cell proliferation comprising, oralternatively consisting of, administering to an animal in which suchinhibition or reduction is desired, a TR21 polypeptide in an amounteffective to inhibit or reduce B cell proliferation.

In a further embodiment, the invention provides methods and compositionsfor stimulating or increasing B cell proliferation, comprising, oralternatively consisting of, contacting an effective amount of TR21polypeptides of the invention (including TR21 fragments and variants,and anti-TR21 antibodies) with Neutrokine-alpha, wherein the effectiveamount of TR21 polypeptide stimulates or increases Neutrokine-alphamediated B cell proliferation.

In another embodiment, the invention provides methods and compositionsfor stimulating or increasing B cell proliferation, comprising, oralternatively consisting of, contacting an effective amount of TR21polypeptides of the invention (including TR21 fragments and variants,and anti-TR21 antibodies) with Neutrokine-alpha SV, wherein theeffective amount of TR21 polypeptide stimulates or increasesNeutrokine-alpha SV mediated B cell proliferation.

In another embodiment, the invention provides methods and compositionsfor stimulating or increasing B cell proliferation, comprising, oralternatively consisting of, contacting an effective amount of TR21polypeptides of the invention (including TR21 fragments and variants,and anti-TR21 antibodies) with one or more heteromultimeric polypeptidecomplexes comprising one or more Neutrokine-alpha and/orNeutrokine-alpha SV polypeptides, wherein the effective amount of TR21polypeptide stimulates or increases B cell proliferation mediated bysaid hetermiultimeric polypeptide complexes.

In another embodiment, the invention provides methods and compositionsfor stimulating or increasing B cell proliferation comprising, oralternatively consisting of, administering to an animal in which suchinhibition or reduction is desired, a TR21 polypeptide in an amounteffective to stimulate or increase B cell proliferation.

In yet another embodiment, the invention provides methods andcompositions for inhibiting or reducing activation of B cells,comprising, or alternatively consisting of, contacting an effectiveamount of TR21 polypeptides of the invention (including TR21 fragmentsand variants, and anti-TR21 antibodies) with Neutrokine-alpha, whereinthe effective amount TR21 polypeptides inhibits or reducesNeutrokine-alpha mediated B cell activation.

In another embodiment, the invention provides methods and compositionsfor inhibiting or reducing activation of B cells, comprising, oralternatively consisting of, contacting an effective amount of TR21polypeptides of the invention (including TR21 fragments and variants,and anti-TR21 antibodies) with Neutrokine-alpha SV, wherein theeffective amount TR21 polypeptides inhibits or reduces Neutrokine-alphaSV mediated B cell activation.

In another embodiment, the invention provides methods and compositionsfor inhibiting or reducing activation of B cells, comprising, oralternatively consisting of, contacting an effective amount of TR21polypeptides of the invention (including TR21 fragments and variants,and anti-TR21 antibodies) with one or more heteromultimeric polypeptidecomplexes comprising one or more Neutrokine-alpha and/orNeutrokine-alpha SV polypeptides, wherein the effective amount TR21polypeptides inhibits or reduces B cell activation mediated by saidheteromultimeric polypeptide complexes.

In another embodiment, the invention provides methods and compositionsfor inhibiting or reducing activation of B cells, comprising, oralternatively consisting of, administering to an animal in which suchinhibition or reduction is desired, a TR21 polypeptide in an amounteffective to inhibit or reduce B cell activation.

In yet another embodiment, the invention provides methods andcompositions for stimulating or increasing activation of B cells,comprising, or alternatively consisting of, contacting an effectiveamount of TR21 polypeptides of the invention (including TR21 fragmentsand variants, and anti-TR21 antibodies) with Neutrokine-alpha, whereinthe effective amount TR21 polypeptides stimulates or increasesNeutrokine-alpha mediated B cell activation.

In another embodiment, the invention provides methods and compositionsfor stimulating or increasing activation of B cells, comprising, oralternatively consisting of, contacting an effective amount of TR21polypeptides of the invention (including TR21 fragments and variants,and anti-TR21 antibodies) with Neutrokine-alpha SV, wherein theeffective amount TR21 polypeptides stimulates or increasesNeutrokine-alpha SV mediated B cell activation.

In another embodiment, the invention provides methods and compositionsfor stimulating or increasing activation of B cells, comprising, oralternatively consisting of, contacting an effective amount of TR21polypeptides of the invention (including TR21 fragments and variants,and anti-TR21 antibodies) with one or more heteromultimeric polypeptidecomplexes comprising one or more Neutrokine-alpha and/orNeutrokine-alpha SV polypeptides, wherein the effective amount TR21polypeptides stimulates or increases B cell activation mediated by saidheteromultimeric polypeptide complexes.

In another embodiment, the invention provides methods and compositionsfor stimulating or increasing activation of B cells, comprising, oralternatively consisting of, administering to an animal in which suchinhibition or reduction is desired, a TR21 polypeptide in an amounteffective to stimulate or increase B cell activation.

B cell activation can measured in a variety of ways, such as FACSanalysis of activation markers expressed on B cells. B cells activationmarkers include, but are not limited to, CD26, CD28, CD30, CD38, CD39,CD69, CD70, CD71, CD 77, CD 83, CD126, CDw130, and B220. Additionally, Bcell activation may be measured by analysis of the activation ofsignaling molecules involved in B cell activation. By way ofnon-limiting example, such analysis may take the form of analyzing mRNAlevels of signaling molecules by Northern analysis or real time PCR. Onecan also measure, for example, the phosphorylation of signalingmolecules using anti-phosphotyrosine antibodies in a Western blot. Bcell activation may also be measured by measuring the calcium levels inB cells. These and other methods of determining B cell activation arecommonly known in the art and could be routinely adapted for the use ofdetermining the effect of TR21 polypeptides of the invention on B cellactivation.

In yet another embodiment, the invention provides methods andcompositions for reducing or decreasing lifespan of B cells, comprising,or alternatively consisting of, contacting an effective amount of TR21polypeptides of the invention (including TR21 fragments and variants,and anti-TR21 antibodies) with Neutrokine-alpha, wherein the effectiveamount of TR21 polypeptide reduces or decreases Neutrokine-alpharegulated lifespan of B cells.

In another embodiment, the invention provides methods and compositionsfor reducing or decreasing lifespan of B cells, comprising, oralternatively consisting of, contacting an effective amount of TR21polypeptides of the invention (including TR21 fragments and variants,and anti-TR21 antibodies) with Neutrokine-alpha SV, wherein theeffective amount of TR21 polypeptide reduces or decreasesNeutrokine-alpha SV regulated lifespan of B cells.

In another embodiment, the invention provides methods and compositionsfor reducing or decreasing lifespan of B cells, comprising, oralternatively consisting of, contacting an effective amount of TR21polypeptides of the invention (including TR21 fragments and variants,and anti-TR21 antibodies) with one or more heteromultimeric polypeptidecomplexes comprising one or more Neutrokine-alpha and/orNeutrokine-alpha SV polypeptides, wherein the effective amount of TR21polypeptide reduces or decreases lifespan of B cells regulated by saidheteromultimeric polypeptide complexes.

In another embodiment, the invention provides methods and compositionsfor reducing or decreasing lifespan of B cells, comprising, oralternatively consisting of, administering to an animal in which suchdecrease is desired, a TR21 polypeptide of the invention in an amounteffective to reduce or decrease B cell lifespan.

In a further embodiment, the invention provides methods and compositionsfor increasing lifespan of B cells, comprising, or alternativelyconsisting of, contacting an effective amount of TR21 polypeptides ofthe invention (including TR21 fragments and variants, and anti-TR21antibodies) with Neutrokine-alpha, wherein the effective amount of TR21polypeptides increases Neutrokine-alpha regulated lifespan of B cells.

In another embodiment, the invention provides methods and compositionsfor increasing lifespan of B cells, comprising, or alternativelyconsisting of, contacting an effective amount of TR21 polypeptides ofthe invention (including TR21 fragments and variants, and anti-TR21antibodies) with Neutrokine-alpha SV, wherein the effective amount ofTR21 polypeptides increases Neutrokine-alpha SV regulated lifespan of Bcells.

In another embodiment, the invention provides methods and compositionsfor increasing lifespan of B cells, comprising, or alternativelyconsisting of, contacting an effective amount of TR21 polypeptides ofthe invention (including TR21 fragments and variants, and anti-TR21antibodies) with one or more heteromultimeric polypeptide complexescomprising one or more Neutrokine-alpha and/or Neutrokine-alpha SVpolypeptides, wherein the effective amount of TR21 polypeptide increaseslifespan of B cells regulated by said heteromultimeric polypeptidecomplexes.

In one embodiment, the invention provides methods and compositions forincreasing lifespan of B cells, comprising, or alternatively consistingof, administering to an animal in which such increase is desired, a TR21polypeptide of the invention (e.g., anti-TR21 antibody) in an amounteffective to increase lifespan of B cells.

B cell lifespan in vivo may be measured by 5-bromo-2′-deoxyuridine(BrdU) labeling experiments which are well known to one skilled in theart. BrdU is a thymidine analogue that gets incorporated into the DNA ofdividing cells. Cells containing BrdU in their DNA can be detectedusing, for example fluorescently labeled anti-BrdU antibody and flowcytometry. Briefly, an animal is injected with BrdU in an amountsufficient to label developing B cells. Then, a sample of B cells iswithdrawn from the animal, for example, from peripheral blood, andanalyzed for the percentage of cells that contain BrdU. Such an analysisperformed at several time points can be used to calculate the halflifeof B cells. Alternatively, B cell survival may be measured in vitro. Forexample, B cells may be cultured under conditions where proliferationdoes not occur, (for example the media should contain no reagents thatcrosslink the immunoglobulin receptor, such as anti-IgM antibodies) fora period of time (usually 2-4 days). At the end of this time, thepercent of surviving cells is determined, using for instance, the vitaldye Trypan Blue, or by staining cells with propidium iodide or any otheragent designed to specifically stain apoptotic cells and analyzing thepercentage of cells stained using flow cytometry. One could perform thisexperiment under several conditions, such as B cells treated withNeutrokine-alpha, B cells treated with Neutrokine-alpha SV, B cellstreated with Neutrokine-alpha/TR21 complexes, B cells treated withNeutrokine-alpha SV/TR21 complexes, and untreated B cells in order todetermine the effects of Neutrokine-alpha, Neutrokine-alpha SV and TR21polypeptides on B cell survival. These and other methods for determiningB cell lifespan are commonly known in the art and could routinely beadapted to determining the effect of TR21 polypeptides onNeutrokine-alpha and/or Neutrokine-alpha SV regulation of B celllifespan.

In one embodiment, the invention provides a method of deliveringcompositions containing the polypeptides of the invention (e.g.,compositions containing TR21 polypeptides or anti-TR21 antibodiesassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs) to targeted cells, such as, for example, monocyticcells expressing TR21 ligand (e.g., Neutrokine-alpha and/orNeutrokine-alpha SV), or B cells expressing TR21. TR21 polypeptides(e.g., soluble TR21 extracellular domain or fragments thereof) oranti-TR21 antibodies of the invention may be associated withheterologous polypeptides, heterologous nucleic acids, toxins, orprodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions.

In one embodiment, the invention provides a method for the specificdelivery of compositions of the invention to cells by administeringpolypeptides of the invention (e.g., TR21 polypeptides or anti-TR21antibodies) that are associated with heterologous polypeptides ornucleic acids. In one example, the invention provides a method fordelivering a therapeutic protein into the targeted cell. In anotherexample, the invention provides a method for delivering a singlestranded nucleic acid (e.g., antisense or ribozymes) or double strandednucleic acid (e.g., DNA that can integrate into the cell's genome orreplicate episomally and that can be transcribed) into the targetedcell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., TR21 polypeptides oranti-TR21 antibodies) in association with toxins or cytotoxic prodrugs.

In a specific embodiment, the invention provides a method for thespecific destruction of cells of monocytic lineage (e.g., monocytic cellrelated leukemias or lymphomas, such as, for example acute myelogenousleukemia) by administering TR21 polypeptides (e.g., a soluble fragmentof the TR21 extracellular domain) and/or anti-TR21 antibodies) inassociation with toxins or cytotoxic prodrugs.

In another specific embodiment, the invention provides a method for thespecific destruction of cells of B cell lineage (e.g., B cell relatedleukemias or lymphomas such as chronic lymphocytic leukemia, multiplemyeloma, non-Hodgkin's lymphoma, and Hodgkin's disease) by administeringanti-TR21 antibodies in association with toxins or cytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, cytotoxins (cytotoxic agents), or anymolecules or enzymes not normally present in or on the surface of a cellthat under defined conditions cause the cell's death. Toxins that may beused according to the methods of the invention include, but are notlimited to, radioisotopes known in the art, compounds such as, forexample, antibodies (or complement fixing containing portions thereof)that bind an inherent or induced endogenous cytotoxic effector system,thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin,Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin,pokeweed antiviral protein, alpha-sarcin and cholera toxin. “Toxin” alsoincludes a cytostatic or cytocidal agent, a therapeutic agent or aradioactive metal ion, e.g., alpha-emitters such as, for example, ²¹³Bi,or other radioisotopes such as, for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se,¹¹³Sn, ⁹⁰Yttrium, ¹¹⁷Tin, ¹⁸⁶Rhenium, ¹⁶⁶Holmium, and ¹⁸⁸Rhenium;luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

Techniques known in the art may be applied to label antibodies of theinvention. Such techniques include, but are not limited to, the use ofbifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065;5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990;5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contentsof each of which are hereby incorporated by reference in its entirety).A cytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

By “cytotoxic prodrug” is meant a non-toxic compound that is convertedby an enzyme, normally present in the cell, into a cytotoxic compound.Cytotoxic prodrugs that may be used according to the methods of theinvention include, but are not limited to, glutamyl derivatives ofbenzoic acid mustard alkylating agent, phosphate derivatives ofetoposide or mitomycin C, cytosine arabinoside, daunorubisin, andphenoxyacetamide derivatives of doxorubicin.

In specific embodiments, TR21 and/or anti-TR21 polypeptides inassociation with radioisotopes, toxins or cytotoxic prodrugs are used totreat or ameliorate the symptoms of autoimmune diseases. In preferredembodiments, TR21 and/or anti-TR21 polypeptides in association withradioisotopes, toxins or cytotoxic prodrugs are used to treat orameliorate the symptoms of systemic lupus erythematosus. In furtherpreferred embodiments, TR21 and/or anti-TR21 polypeptides in associationwith radioisotopes, toxins or cytotoxic prodrugs are used to treat orameliorate the symptoms of rheumatoid arthritis, including advancedrheumatoid arthritis. In preferred embodiments, TR21 and/or anti-TR21polypeptides in association with radioisotopes, toxins or cytotoxicprodrugs are used to treat or ameliorate the symptoms of idiopathicthrombocytopenic purpura (ITP).

In other preferred embodiments TR21 and/or anti-TR21 polypeptides inassociation with radioisotopes, toxins or cytotoxic prodrugs are used totreat or ameliorate the symptoms of Sjögren's syndrome. In otherpreferred embodiments, TR21 and/or anti-TR21 polypeptides in associationwith radioisotopes, toxins or cytotoxic prodrugs are used to treat orameliorate the symptoms of IgA nephropathy. In other preferredembodiments, TR21 and/or anti-TR21 polypeptides in association withradioisotopes, toxins or cytotoxic prodrugs are used to treat orameliorate the symptoms of Myasthenia gravis. In preferred embodiments,TR21 and/or anti-TR21 polypeptides in association with radioisotopes,toxins or cytotoxic prodrugs are used to treat or ameliorate thesymptoms of multiple sclerosis.

It will be appreciated that conditions caused by a decrease in thestandard or normal level of TR21 activity or TR21 ligand (e.g.,Neutrokine-alpha) activity in an individual, particularly disorders ofthe immune system (e.g., an immunodeficiency), can be treated byadministration of TR21 polypeptide (e.g., in the form of solubleextracellular domain or fragments thereof, or cells expressing thecomplete protein) or agonist. Thus, the invention also provides a methodof treatment of an individual in need of an increased level of TR21activity or TR21 ligand activity comprising administering to such anindividual a pharmaceutical composition comprising an amount of anisolated TR21 polypeptide of the invention, or agonist thereof,effective to increase the TR21 activity level in such an individual.

It will also be appreciated that conditions caused by a increase in thestandard or normal level of TR21 activity or TR21 ligand (e.g.,Neutrokine-alpha) activity in an individual, particularly disorders ofthe immune system (e.g., autoimmune diseases, such as, for example,lupus, rheumatoid arthritis, and myasthenia gravis), can be treated byadministration of TR21 polypeptides (e.g., in the form of solubleextracellular domain or fragments thereof, or cells expressing thecomplete protein) or antagonist (e.g., an anti-TR21 antibody). Thus, theinvention also provides a method of treatment of an individual in needof a decreased level of TR21 activity or TR21 ligand activity comprisingadministering to such an individual a pharmaceutical compositioncomprising an amount of an isolated TR21 polypeptide of the invention,or antagonist thereof, effective to decrease the TR21 activity level insuch an individual.

Autoantibody production is common to several autoimmune diseases andcontributes to tissue destruction and exacerbation of disease.Autoantibodies can also lead to the occurrence of immune complexdeposition complications and lead to many symptoms of systemic lupuserythematosis, including kidney failure, neuralgic symptoms and death.Modulating antibody production independent of cellular response wouldalso be beneficial in many disease states. B cells have also been shownto play a role in the secretion of arthritogenic immunoglobulins inrheumatoid arthritis, (Korganow et al., Immunity 10:451-61, 1999). Assuch, inhibition of Neutrokine-alpha mediated antibody production wouldbe beneficial in treatment of autoimmune diseases such as myastheniagravis and rheumatoid arthritis. Compounds of the invention thatselectively block or neutralize the action of B-lymphocytes would beuseful for such purposes. To verify these capabilities in compositionsof the present invention, such compositions are evaluated using assaysknown in the art and described herein.

The invention provides methods employing compositions of the invention(e.g., TR21 polynucleotides or polypeptides of the invention and/oragonists and/or antagonists thereof) for selectively blocking orneutralizing the actions of B-cells in association with end stage renaldiseases, which may or may not be associated with autoimmune diseases.Such methods would also be useful for treating immunologic renaldiseases. Such methods would be would be useful for treatingglomerulonephritis associated with diseases such as membranousnephropathy, IgA nephropathy or Berger's Disease, IgM nephropathy,Goodpasture's Disease, post-infectious glomerulonephritis,mesangioproliferative disease, minimal-change nephrotic syndrome. Suchmethods would also serve as therapeutic applications for treatingsecondary glomerulonephritis or vasculitis associated with such diseasesas lupus, polyarteritis, Henloch-Scoenlein, Scleroderma, HIV-relateddiseases, amyloidosis or hemolytic uremic syndrome. The methods of thepresent invention would also be useful as part of a therapeuticapplication for treating interstitial nephritis or pyelonephritisassociated with chronic pyelonephritis, analgesic abuse,nephrocalcinosis, nephropathy caused by other agents, nephrolithiasis,or chronic or acute interstitial nephritis.

The methods of the present invention also include use of compositions ofthe invention in the treatment of hypertensive or large vessel diseases,including renal artery stenosis or occlusion and cholesterol emboli orrenal emboli.

The present invention also provides methods for diagnosis and treatmentof renal or urological neoplasms, multiple mylelomas, lymphomas, lightchain neuropathy or amyloidosis.

The invention also provides methods for blocking or inhibiting activatedB cells using compositions of the invention for the treatment of asthmaand other chronic airway diseases such as bronchitis and emphysema.

TR21 polynucleotides or polypeptides of the invention, or agonists ofTR21 (e.g., anti-TR21 agonistic antibodies), can be used in thetreatment of infectious agents. For example, by increasing the immuneresponse, particularly increasing the proliferation and differentiationof B cells, infectious diseases may be treated. The immune response maybe increased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively TR21 polynucleotides orpolypeptides of the invention, or agonists of TR21 (e.g., anti-TR21agonistic antibodies), may also directly inhibit the infectious agent,without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated, prevented, and/or diagnosed by TR21polynucleotides or polypeptides of the invention, or agonists of TR21(e.g., anti-TR21 agonistic antibodies). Examples of viruses, that can betreated, prevented, and/or diagnosed with the compositions of theinvention include, but are not limited to one or more of the followingDNA and RNA viruses and viral families: Arbovirus, Adenoviridae,Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae,Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae,Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus,Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae,Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A,Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia),Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling withinthese families can cause a variety of diseases or symptoms, including,but not limited to: arthritis, bronchiollitis, respiratory syncytialvirus, encephalitis, eye infections (e.g., conjunctivitis, keratitis),chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta),Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellowfever, meningitis, opportunistic infections (e.g., AIDS), pneumonia,Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia. TR21 polynucleotides or polypeptides, or agonists orantagonists of TR21, can be used to treat, prevent, diagnose, and/ordetect any of these symptoms or diseases. In specific embodiments, TR21polynucleotides or polypeptides, or agonists of TR21 are used to treat,prevent, and/or diagnose: meningitis, Dengue, EBV, and/or hepatitis(e.g., hepatitis B). In an additional specific embodiment TR21polynucleotides, polypeptides, or agonists are used to treat patientsnonresponsive to one or more other commercially available hepatitisvaccines. In a further specific embodiment, TR21 polynucleotides,polypeptides, or agonists are used to treat, prevent, and/or diagnoseAIDS. In an additional specific embodiment TR21 polynucleotides,polypeptides, agonists, and/or antagonists are used to treat, prevent,and/or diagnose patients with cryptosporidiosis.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated, prevented, and/or diagnosed by TR21polynucleotides or polypeptides, or agonists or antagonists of TR21,include, but not limited to, one or more of the following Gram-Negativeand Gram-positive bacteria and bacterial families and fungi:Actinomycetales (e.g., Corynebacterium, Mycobacterium, Nocardia),Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax,Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g.,Borrelia burgdorferi, Brucellosis, Candidiasis, Campylobacter,Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E. coli (e.g.,Enterotoxigenic E. coli and Enterohemorrhagic E. coli),Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, andSalmonella paratyphi), Serratia, Yersinia), Erysipelothrix,Helicobacter, Legionellosis, Leptospirosis, Listeria (e.g., Listeriamonocytogenes), Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae,Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Neisseriameningitidis, Pasteurellacea Infections (e.g., Actinobacillus,Haemophilus (e.g., Haemophilus influenza type B), Pasteurella),Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp.,Staphylococcal, Meningococcal, Pneumococcal and Streptococcal (e.g.,Streptococcus pneumoniae and Group B Streptococcus). These bacterial orfungal families can cause the following diseases or symptoms, including,but not limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., meningitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. TR21 polynucleotides or polypeptides, or agonists orantagonists of TR21, can be used to treat, prevent, diagnose, and/ordetect any of these symptoms or diseases. In specific embodiments, TR21polynucleotides, polypeptides, or agonists thereof are used to treat,prevent, and/or diagnose: tetanus, Diphtheria, botulism, and/ormeningitis type B.

Moreover, parasitic agents causing disease or symptoms that can betreated, prevented, and/or diagnosed by TR21 polynucleotides orpolypeptides, or agonists or antagonists of TR21, include, but notlimited to, a member of one or more of the following families or class:Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis,Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis,Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas andSporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodiummalariae and Plasmodium ovale). These parasites can cause a variety ofdiseases or symptoms, including, but not limited to: Scabies,Trombiculiasis, eye infections, intestinal disease (e.g., dysentery,giardiasis), liver disease, lung disease, opportunistic infections(e.g., AIDS related), malaria, pregnancy complications, andtoxoplasmosis. TR21 polynucleotides or polypeptides, or agonists orantagonists of TR21, can be used to treat, prevent, diagnose, and/ordetect any of these symptoms or diseases. In specific embodiments, TR21polynucleotides, polypeptides, or agonists thereof are used to treat,prevent, and/or diagnose malaria.

In another embodiment, TR21 polynucleotides or polypeptides of theinvention and/or agonists and/or antagonists thereof, are used to treat,prevent, and/or diagnose inner ear infection (such as, for example,otitis media), as well as other infections characterized by infectionwith Streptococcus pneumoniae and other pathogenic organisms.

In a specific embodiment, TR21 polynucleotides or polypeptides, oragonists or antagonists thereof (e.g., anti-TR21 antibodies) are used totreat or prevent a disorder characterized by deficient serumimmunoglobulin production, recurrent infections, and/or immune systemdysfunction. Moreover, TR21 polynucleotides or polypeptides, or agonistsor antagonists thereof (e.g., anti-TR21 antibodies) may be used to treator prevent infections of the joints, bones, skin, and/or parotid glands,blood-borne infections (e.g., sepsis, meningitis, septic arthritis,and/or osteomyelitis), autoimmune diseases (e.g., those disclosedherein), inflammatory disorders, and malignancies, and/or any disease ordisorder or condition associated with these infections, diseases,disorders and/or malignancies) including, but not limited to, CVID,other primary immune deficiencies, HIV disease, CLL, multiple myeloma,recurrent bronchitis, sinusitis, otitis media, conjunctivitis,pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpeszoster), and/or pheumocystis carnii.

TR21 polynucleotides or polypeptides of the invention, or agonists orantagonists thereof, may be used to diagnose, prognose, treat or preventone or more of the following diseases or disorders, or conditionsassociated therewith: primary immuodeficiencies, immune-mediatedthrombocytopenia, Kawasaki syndrome, bone marrow transplant (e.g.,recent bone marrow transplant in adults or children), chronic B-celllymphocytic leukemia, HIV infection (e.g., adult or pediatric HIVinfection), chronic inflammatory demyelinating polyneuropathy, andpost-transfusion purpura.

Additionally, TR21 polynucleotides or polypeptides of the invention, oragonists or antagonists thereof, may be used to diagnose, prognose,treat or prevent one or more of the following diseases, disorders, orconditions associated therewith, Guillain-Barre syndrome, anemia (e.g.,anemia associated with parvovirus B19, stable mutliple myeloma with highrisk for infection (e.g., recurrent infection), autoimmune hemolyticanemia (e.g., warm-type autoimmune hemolytic anemia), thrombocytopenia(e.g., neonatal thrombocytopenia), and immune-mediated neutropenia),infection through transplantation (e.g., cytomegalovirus (CMV)-negativerecipients of CMV-positive organs), hypogammaglobulinemia (e.g.,hypogammaglobulinemic neonates with risk factor for infection ormorbidity), epilepsy (e.g., intractable epilepsy), systemic vasculiticsyndromes, myasthenia gravis (e.g., decompensation in myastheniagravis), dermatomyositis, and polymyositis.

Additional preferred embodiments of the invention include, but are notlimited to, the use of TR21 polypeptides, TR21 polynucleotides, andfunctional agonists or antagonists thereof, (e.g., example an anti-TR21agonistic antibody) in the following applications:

Administration to an animal (e.g., mouse, rat, rabbit, hamster, guineapig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat,non-human primate, and human, most preferably human) to boost the immunesystem to produce increased quantities of one or more antibodies (e.g.,IgG, IgA, IgM, and IgE), to induce higher affinity antibody production(e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.In a specific nonexclusive embodiment, TR21 polypeptides of theinvention, and/or agonists thereof, are administered to boost the immunesystem to produce increased quantities of IgG. In another specificnonexclusive embodiment, TR21 polypeptides of the invention and/oragonists thereof, are administered to boost the immune system to produceincreased quantities of IgA. In another specific nonexclusiveembodiment, TR21 polypeptides of the invention and/or agonists thereof,are administered to boost the immune system to produce increasedquantities of IgM.

Administration to an animal (including, but not limited to, those listedabove, and also including transgenic animals) incapable of producingfunctional endogenous antibody molecules or having an otherwisecompromised endogenous immune system, but which is capable of producinghuman immunoglobulin molecules by means of a reconstituted or partiallyreconstituted immune system from another animal (see, e.g., publishedPCT Application Nos. WO98/24893, WO/9634096, WO/9633735, andWO/9110741).

A vaccine adjuvant that enhances immune responsiveness to specificantigen. In a specific embodiment, the vaccine is a TR21 polypeptidedescribed herein. In another specific embodiment, the vaccine adjuvantis a polynucleotide described herein (e.g., a TR21 polynucleotidegenetic vaccine adjuvant). As discussed herein, TR21 polynucleotides maybe administered using techniques known in the art, including but notlimited to, liposomal delivery, recombinant vector delivery, injectionof naked DNA, and gene gun delivery.

An adjuvant to enhance tumor-specific immune responses.

An adjuvant to enhance anti-viral immune responses. Anti-viral immuneresponses that may be enhanced using the compositions of the inventionas an adjuvant, include, but are not limited to, virus and virusassociated diseases or symptoms described herein or otherwise known inthe art. In specific embodiments, the compositions of the invention areused as an adjuvant to enhance an immune response to a virus, disease,or symptom selected from the group consisting of: AIDS, meningitis,Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specificembodiment, the compositions of the invention are used as an adjuvant toenhance an immune response to a virus, disease, or symptom selected fromthe group consisting of: HIV/AIDS, Respiratory syncytial virus, Dengue,Rotavirus, Japanese B encephalitis, Influenza A and B, Parainfluenza,Measles, Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever,Herpes simplex, and yellow fever. In another specific embodiment, thecompositions of the invention are used as an adjuvant to enhance animmune response to the HIV gp120 antigen.

An adjuvant to enhance anti-bacterial or anti-fungal immune responses.Anti-bacterial or anti-fungal immune responses that may be enhancedusing the compositions of the invention as an adjuvant, include bacteriaor fungus and bacteria or fungus associated diseases or symptomsdescribed herein or otherwise known in the art. In specific embodiments,the compositions of the invention are used as an adjuvant to enhance animmune response to a bacterium or fungus, disease, or symptom selectedfrom the group consisting of: tetanus, Diphtheria, botulism, andmeningitis type B. In another specific embodiment, the compositions ofthe invention are used as an adjuvant to enhance an immune response to abacteria or fungus, disease, or symptom selected from the groupconsisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi,Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae,Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli,Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium(malaria).

An adjuvant to enhance anti-parasitic immune responses. Anti-parasiticimmune responses that may be enhanced using the compositions of theinvention as an adjuvant, include parasite and parasite associateddiseases or symptoms described herein or otherwise known in the art. Inspecific embodiments, the compositions of the invention are used as anadjuvant to enhance an immune response to a parasite. In anotherspecific embodiment, the compositions of the invention are used as anadjuvant to enhance an immune response to Plasmodium falciparum(malaria).

As a stimulator of B cell responsiveness to pathogens.

As an agent that elevates the immune status of an individual prior totheir receipt of immunosuppressive therapies.

As an agent to induce higher affinity antibodies.

As an agent to increase serum immunoglobulin concentrations.

As an agent to accelerate recovery of immunocompromised individuals.

As an agent to boost immunoresponsiveness among aged populations.

As an immune system enhancer prior to, during, or after bone marrowtransplant and/or other transplants (e.g., allogenic or xenogenic organtransplantation). With respect to transplantation, compositions of theinvention may be administered prior to, concomitant with, and/or aftertransplantation. In a specific embodiment, compositions of the inventionare administered after transplantation, prior to the beginning ofrecovery of T-cell populations. In another specific embodiment,compositions of the invention are first administered aftertransplantation after the beginning of recovery of T cell populations,but prior to full recovery of B cell populations.

As an agent to boost immunoresponsiveness among B cell immunodeficientindividuals, such as, for example, an individual who has undergone apartial or complete splenectomy. B cell immunodeficiencies that may beameliorated or treated by administering the TR21 polypeptides orpolynucleotides of the invention, or agonists thereof, include, but arenot limited to, severe combined immunodeficiency (SCID)-X linked,SCID-autosomal, adenosine deaminase deficiency (ADA deficiency),X-linked agammaglobulinemia (XLA), Bruton's disease, congenitalagammaglobulinemia, X-linked infantile agammaglobulinemia, acquiredagammaglobulinemia, adult onset agammaglobulinemia, late-onsetagammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,transient hypogammaglobulinemia of infancy, unspecifiedhypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell lymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, severe congenitalleukopenia, thymic alymphoplasia-aplasia or dysplasia withimmunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linkedlymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency.

In a specific embodiment, TR21 polypeptides or polynucleotides of theinvention, or agonists thereof, is administered to treat or ameliorateselective IgA deficiency.

In another specific embodiment, TR21 polypeptides or polynucleotides ofthe invention, or agonists thereof, is administered to treat orameliorate ataxia-telangiectasia.

In another specific embodiment, TR21 polypeptides or polynucleotides ofthe invention, or agonists thereof, is administered to treat orameliorate common variable immunodeficiency.

In another specific embodiment, TR21 polypeptides or polynucleotides ofthe invention, or agonists thereof, is administered to treat orameliorate X-linked agammaglobulinemia.

In another specific embodiment, TR21 polypeptides or polynucleotides ofthe invention, or agonists thereof, is administered to treat orameliorate severe combined immunodeficiency (SCID).

In another specific embodiment, TR21 polypeptides or polynucleotides ofthe invention, or agonists thereof, is administered to treat orameliorate Wiskott-Aldrich syndrome.

In another specific embodiment, TR21 polypeptides or polynucleotides ofthe invention, or agonists thereof, is administered to treat orameliorate X-linked Ig deficiency with hyper IgM.

As an agent to boost immunoresponsiveness among individuals having anacquired loss of B cell function. Conditions resulting in an acquiredloss of B cell function that may be ameliorated or treated byadministering TR21 polypeptides or polynucleotides of the invention, oragonists thereof include, but are not limited to, HIV Infection, AIDS,bone marrow transplant, multiple myeloma and B cell chronic lymphocyticleukemia (CLL).

Patients with CLL and myeloma are at risk for increased infections.Thus, one aspect of the present invention provides for the use of TR21and/or anti-TR21 polynucleotides and/or polypeptides as an agent toboost immunoresponsiveness in CLL and myeloma patients.

As an agent to boost immunoresponsiveness among individuals having atemporary immune deficiency or compromised immune system. Conditionsresulting in a temporary immune deficiency/compromised immune systemthat may be ameliorated or treated by administering TR21 polypeptides orpolynucleotides of the invention, or agonists or antagonists thereof,include, but are not limited to, recovery from viral infections (e.g.,influenza), conditions associated with malnutrition, recovery frominfectious mononucleosis, or conditions associated with stress, recoveryfrom measles, recovery from blood transfusion, recovery from surgery andrecovery from burns.

As a regulator of antigen presentation by monocytes, dendritic cells, Tcells and/or B-cells. In one embodiment, TR21 polypeptides (in soluble,membrane-bound or transmembrane forms) or polynucleotides enhanceantigen presentation or antagonize antigen presentation in vitro or invivo. Moreover, in related embodiments, this enhancement or antagonismof antigen presentation may be useful in anti-tumor treatment or tomodulate the immune system.

As a mediator of mucosal immune responses. The expression ofNeutrokine-alpha on monocytes, the expression of TR21 on B cells, andthe responsiveness of B cells to Neutrokine-alpha suggests that it maybe involved in exchange of signals between B cells and monocytes ortheir differentiated progeny. This activity is in many ways analogous tothe CD40-CD154 signaling between B cells and T cells. TR21 may thereforebe an important regulator of T cell independent immune responses toenvironmental pathogens. In particular, the unconventional B cellpopulations (CD5+) that are associated with mucosal sites andresponsible for much of the innate immunity in humans may respond toTR21 thereby enhancing an individual's protective immune status.

As an agent to direct an individual's immune system towards developmentof a humoral response (i.e. TH2) as opposed to a TH1 cellular response.

As a means to induce tumor proliferation and thus make it moresusceptible to anti-neoplastic agents. For example, multiple myeloma isa slowly dividing disease and is thus refractory to virtually allanti-neoplastic regimens. If these cells were forced to proliferate morerapidly their susceptibility profile would likely change.

As a monocyte cell specific binding protein to which specific activatorsor inhibitors of cell growth may be attached. The result would be tofocus the activity of such activators or inhibitors onto normal,diseased, or neoplastic B cell populations.

As a B cell specific binding protein to which specific activators orinhibitors of cell growth may be attached. The result would be to focusthe activity of such activators or inhibitors onto normal, diseased, orneoplastic B cell populations.

As a means of detecting B-lineage cells by virtue of its specificity.This application may require labeling the protein with biotin or otheragents (e.g., as described herein) to afford a means of detection.

As a stimulator of B cell production in pathologies such as AIDS,chronic lymphocyte disorder and/or Common Variable Immunodeficiency.

As part of a B cell selection device the function of which is to isolateB cells from a heterogeneous mixture of cell types. TR21 could becoupled to a solid support to which B cells would then specificallybind. Unbound cells would be washed out and the bound cells subsequentlyeluted. A nonlimiting use of this selection would be to allow purging oftumor cells from, for example, bone marrow or peripheral blood prior totransplant.

As a therapy for generation and/or regeneration of lymphoid tissuesfollowing surgery, trauma or genetic defect.

As a gene-based therapy for genetically inherited disorders resulting inimmuno-incompetence such as observed among SCID patients.

As an antigen for the generation of antibodies to inhibit or enhanceTR21 mediated responses.

As a means of activating monocytes/macrophages to defend againstparasitic diseases that effect monocytes such as Leishmaniasis.

As pretreatment of bone marrow samples prior to transplant. Suchtreatment would increase B cell representation and thus acceleraterecovery.

As a means of regulating secreted cytokines that are elicited by TR21.For example, as a means of regulating secreted cytokines that areelicited by TR21.

TR21 polypeptides or polynucleotides of the invention, or agonists maybe used to modulate IgE concentrations in vitro or in vivo.

Additionally, TR21 polypeptides or polynucleotides of the invention, oragonists thereof, may be used to treat, prevent, and/or diagnoseIgE-mediated allergic reactions. Such allergic reactions include, butare not limited to, asthma, rhinitis, and eczema.

In a specific embodiment, TR21 polypeptides or polynucleotides of theinvention, or agonists thereof, is administered to treat, prevent,diagnose, and/or ameliorate selective IgA deficiency.

In another specific embodiment, TR21 polypeptides or polynucleotides ofthe invention, or agonists thereof, is administered to treat, prevent,diagnose, and/or ameliorate ataxia-telangiectasia.

In another specific embodiment, TR21 polypeptides or polynucleotides ofthe invention, or agonists thereof, is administered to treat, prevent,diagnose, and/or ameliorate common variable immunodeficiency.

In another specific embodiment, TR21 polypeptides or polynucleotides ofthe invention, or agonists thereof, is administered to treat, prevent,diagnose, and/or ameliorate X-linked agammaglobulinemia.

In another specific embodiment, TR21 polypeptides or polynucleotides ofthe invention, or agonists thereof, is administered to treat, prevent,diagnose, and/or ameliorate severe combined immunodeficiency (SCID).

In another specific embodiment, TR21 polypeptides or polynucleotides ofthe invention, or agonists thereof, is administered to treat, prevent,diagnose, and/or ameliorate Wiskott-Aldrich syndrome.

In another specific embodiment, TR21 polypeptides or polynucleotides ofthe invention, or agonists thereof, is administered to treat, prevent,diagnose, and/or ameliorate X-linked Ig deficiency with hyper IgM. In aspecific embodiment, TR21 polypeptides or polynucleotides of theinvention, or agonists thereof, is administered to treat, prevent,diagnose, and/or ameliorate X-linked Ig deficiency with hyper IgM.

In another specific embodiment, TR21 polypeptides or polynucleotides ofthe invention, or agonists or antagonists (e.g., anti-TR21 antibodies)thereof, is administered to treat, prevent, and/or diagnose chronicmyelogenous leukemia, acute myelogenous leukemia, leukemia, hystiocyticleukemia, monocytic leukemia (e.g., acute monocytic leukemia), leukemicreticulosis, Shilling Type monocytic leukemia, and/or other leukemiasderived from monocytes and/or monocytic cells and/or tissues.

In another specific embodiment, TR21 polypeptides or polynucleotides ofthe invention, or agonists thereof, is administered to treat, prevent,diagnose, and/or ameliorate monocytic leukemoid reaction, as seen, forexample, with tuberculosis.

In another specific embodimentTR21 polypeptides or polynucleotides ofthe invention, or agonists thereof, is administered to treat, prevent,diagnose, and/or ameliorate monocytic leukocytosis, monocyticleukopenia, monocytopenia, and/or monocytosis.

In a specific embodiment, TR21 polynucleotides or polypeptides of theinvention, and/or anti-TR21 antibodies, and/or agonists or antagoniststhereof, are used to treat, prevent, detect, and/or diagnose primary Blymphocyte disorders and/or diseases, and/or conditions associatedtherewith. In one embodiment, such primary B lymphocyte disorders,diseases, and/or conditions are characterized by a complete or partialloss of humoral immunity. Primary B lymphocyte disorders, diseases,and/or conditions associated therewith that are characterized by acomplete or partial loss of humoral immunity and that may be prevented,treated, detected and/or diagnosed with compositions of the inventioninclude, but are not limited to, X-Linked Agammaglobulinemia (XLA),severe combined immunodeficiency disease (SCID), and selective IgAdeficiency.

In a preferred embodiment, TR21 polynucleotides, polypeptides, and/oragonists and/or antagonists thereof are used to treat, prevent, and/ordiagnose diseases or disorders affecting or conditions associated withany one or more of the various mucous membranes of the body. Suchdiseases or disorders include, but are not limited to, for example,mucositis, mucoclasis, mucocolitis, mucocutaneous leishmaniasis (suchas, for example, American leishmaniasis, leishmaniasis americana,nasopharyngeal leishmaniasis, and New World leishmaniasis),mucocutaneous lymph node syndrome (for example, Kawasaki disease),mucoenteritis, mucoepidermoid carcinoma, mucoepidermoid tumor,mucoepithelial dysplasia, mucoid adenocarcinoma, mucoid degeneration,myxoid degeneration; myxomatous degeneration; myxomatosis, mucoid medialdegeneration (for example, cystic medial necrosis), mucolipidosis(including, for example, mucolipidosis I, mucolipidosis II,mucolipidosis III, and mucolipidosis IV), mucolysis disorders,mucomembranous enteritis, mucoenteritis, mucopolysaccharidosis (such as,for example, type I mucopolysaccharidosis (i.e., Hurler's syndrome),type IS mucopolysaccharidosis (i.e., Scheie's syndrome or type Vmucopolysaccharidosis), type II mucopolysaccharidosis (i.e., Hunter'ssyndrome), type III mucopolysaccharidosis (i.e., Sanfilippo's syndrome),type IV mucopolysaccharidosis (i.e., Morquio's syndrome), type VImucopolysaccharidosis (i.e., Maroteaux-Lamy syndrome), type VIImucopolysaccharidosis (i.e, mucopolysaccharidosis due tobeta-glucuronidase deficiency), and mucosulfatidosis),mucopolysacchariduria, mucopurulent conjunctivitis, mucopus,mucormycosis (i.e., zygomycosis), mucosal disease (i.e., bovine virusdiarrhea), mucous colitis (such as, for example, mucocolitis andmyxomembranous colitis), and mucoviscidosis (such as, for example,cystic fibrosis, cystic fibrosis of the pancreas, Clarke-Hadfieldsyndrome, fibrocystic disease of the pancreas, mucoviscidosis, andviscidosis). In a highly preferred embodiment, TR21 polynucleotides,polypeptides, and/or agonists and/or antagonists thereof are used totreat, prevent, and/or diagnose mucositis, especially as associated withchemotherapy.

In a preferred embodiment, TR21 polynucleotides, polypeptides, and/oragonists and/or antagonists thereof are used to treat, prevent, and/ordiagnose diseases or disorders affecting or conditions associated withsinusitis.

An additional condition, disease or symptom that can be treated,prevented, and/or diagnosed by TR21 polynucleotides or polypeptides, oragonists of TR21 (e.g., anti-TR21 agonistic antibodies), isosteomyelitis.

An additional condition, disease or symptom that can be treated,prevented, and/or diagnosed by TR21 polynucleotides or polypeptides, oragonists of TR21 (e.g., anti-TR21 agonistic antibodies), isendocarditis.

All of the above described applications as they may apply to veterinarymedicine.

Antagonists of TR21 include binding and/or inhibitory antibodies (e.g.,anti-TR21 antagonistic antibodies), antisense nucleic acids, ribozymes,and TR21 polypeptides of the invention. These would be expected toreverse many of the activities of the ligand described above as well asfind clinical or practical application as:

A means of blocking various aspects of immune responses to foreignagents or self. Examples include autoimmune disorders such as lupus, andarthritis, as well as immunoresponsiveness to skin allergies,inflammation, bowel disease, injury and pathogens. Although thereappears to be a clear potential role of TR21 in B cell and monocyterelated pathologies, it remains possible that other cell types may gainexpression or responsiveness to TR21. Thus, TR21 may, like CD40 and itsligand, be regulated by the status of the immune system and themicroenvironment in which the cell is located.

A therapy for preventing the B cell proliferation and Ig secretionassociated with autoimmune diseases such as idiopathic thrombocytopenicpurpura, systemic lupus erythematosus and MS.

An inhibitor of graft versus host disease or transplant rejection.

A therapy for B cell malignancies such as ALL, Hodgkin's disease,non-Hodgkin's lymphoma, Chronic lymphocyte leukemia, plasmacytomas,multiple myeloma, Burkitt's lymphoma, and EBV-transformed diseases.

A therapy for chronic hypergammaglobulinemeia evident in such diseasesas monoclonalgammopathy of undetermined significance (MGUS),Waldenstrom's disease, related idiopathic monoclonalgammopathies, andplasmacytomas.

A therapy for decreasing cellular proliferation of Large B-cellLymphomas.

A means of decreasing the involvement of B cells and Ig associated withChronic Myelogenous Leukemia.

As a B cell specific binding protein to which specific activators orinhibitors of cell growth may be attached. The result would be to focusthe activity of such activators or inhibitors onto normal, diseased, orneoplastic B cell populations.

As part of a B cell selection device the function of which is to isolateB cells from a heterogeneous mixture of cell types. Anti-TR21 antibodyor TNF ligands that bind TR21 could be coupled to a solid support towhich B cells would then specifically bind. Unbound cells would bewashed out and the bound cells subsequently eluted. This technique wouldallow purging of tumor cells from, for example, bone marrow orperipheral blood prior to transplant.

An immunosuppressive agent(s).

TR21 polypeptides or polynucleotides of the invention, or antagonistsmay be used to modulate IgE concentrations in vitro or in vivo.

In another embodiment, administration of TR21 polypeptides orpolynucleotides of the invention, or antagonists thereof, may be used totreat, prevent, and/or diagnose IgE-mediated allergic reactionsincluding, but not limited to, asthma, rhinitis, and eczema.

An inhibitor of signaling pathways involving ERK1, COX2 and Cyclin D2.

The above-recited applications have uses in a wide variety of hosts.Such hosts include, but are not limited to, human, murine, rabbit, goat,guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken,goat, cow, sheep, dog, cat, non-human primate, and human. In specificembodiments, the host is a mouse, rabbit, goat, guinea pig, chicken,rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the hostis a mammal. In most preferred embodiments, the host is a human.

The agonists and antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as described herein.

The antagonists may be employed, for instance, to inhibit TR21-mediatedchemotaxis and activation of macrophages and their precursors, and ofneutrophils, basophils, B lymphocytes and some T-cell subsets, e.g.,activated and CD8 cytotoxic T cells and natural killer cells, in certainauto-immune and chronic inflammatory and infective diseases. Examples ofauto-immune diseases include multiple sclerosis, and insulin-dependentdiabetes. The antagonists may also be employed to treat, prevent, and/ordiagnose infectious diseases including silicosis, sarcoidosis,idiopathic pulmonary fibrosis by preventing the recruitment andactivation of mononuclear phagocytes. They may also be employed totreat, prevent, and/or diagnose idiopathic hyper-eosinophilic syndromeby preventing eosinophil production and migration. Endotoxic shock mayalso be treated by the antagonists by preventing the migration ofmacrophages and their production of the TR21 polypeptides of the presentinvention. The antagonists may also be employed for treatingatherosclerosis, by preventing monocyte infiltration in the artery wall.The antagonists may also be employed to treat, prevent, and/or diagnosehistamine-mediated allergic reactions and immunological disordersincluding late phase allergic reactions, chronic urticaria, and atopicdermatitis by inhibiting chemokine-induced mast cell and basophildegranulation and release of histamine. IgE-mediated allergic reactionssuch as allergic asthma, rhinitis, and eczema may also be treated. Theantagonists may also be employed to treat, prevent, and/or diagnosechronic and acute inflammation by preventing the attraction of monocytesto a wound area. They may also be employed to regulate normal pulmonarymacrophage populations, since chronic and acute inflammatory pulmonarydiseases are associated with sequestration of mononuclear phagocytes inthe lung. Antagonists may also be employed to treat, prevent, and/ordiagnose rheumatoid arthritis by preventing the attraction of monocytesinto synovial fluid in the joints of patients. Monocyte influx andactivation plays a significant role in the pathogenesis of bothdegenerative and inflammatory arthropathies. The antagonists may beemployed to interfere with the deleterious cascades attributed primarilyto IL-1 and TNF, which prevents the biosynthesis of other inflammatorycytokines. In this way, the antagonists may be employed to preventinflammation. The antagonists may also be employed to inhibitprostaglandin-independent fever induced by TR21. The antagonists mayalso be employed to treat, prevent, and/or diagnose cases of bone marrowfailure, for example, aplastic anemia and myelodysplastic syndrome. Theantagonists may also be employed to treat, prevent, and/or diagnoseasthma and allergy by preventing eosinophil accumulation in the lung.The antagonists may also be employed to treat, prevent, and/or diagnosesubepithelial basement membrane fibrosis which is a prominent feature ofthe asthmatic lung. The antagonists may also be employed to treat,prevent, and/or diagnose lymphomas (e.g., one or more of the extensive,but not limiting, list of lymphomas provided herein).

All of the above described applications as they may apply to veterinarymedicine. Moreover, all applications described herein may also apply toveterinary medicine.

TR21 polynucleotides or polypeptides of the invention and/or agonistsand/or antagonists thereof, may be used to treat, prevent, and/ordiagnose various immune system-related disorders and/or conditionsassociated with these disorders, in mammals, preferably humans. Manyautoimmune disorders result from inappropriate recognition of self asforeign material by immune cells. This inappropriate recognition resultsin an immune response leading to the destruction of the host tissue.Therefore, the administration of TR21 polynucleotides or polypeptides ofthe invention and/or agonists and/or antagonists thereof that caninhibit an immune response, particularly the proliferation of B cellsand/or the production of immunoglobulins, may be an effective therapy intreating and/or preventing autoimmune disorders. Thus, in preferredembodiments, TR21 polypeptides and/or TR21 antagonists of the invention(e.g., polypeptide fragments of TR21 and anti-TR21 antibodies) are usedto treat, prevent, and/or diagnose an autoimmune disorder.

Autoimmune disorders and conditions associated with these disorders thatmay be treated, prevented, and/or diagnosed with the TR21polynucleotides, polypeptides, and/or antagonist (e.g., anti-TR21antibodies) of the invention, include, but are not limited to,autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia,idiopathic thrombocytopenia purpura, autoimmune neutropenia,autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,dermatitis, gluten-sensitive enteropathy, allergic encephalomyelitis,myocarditis, relapsing polychondritis, rheumatic heart disease,glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis,Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g.,Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome,Autoimmune Pulmonary Inflammation, myocarditis, IgA glomerulonephritis,dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome,insulin dependent diabetes mellitis, and autoimmune inflammatory eyedisease.

Additional autoimmune disorders (that are highly probable) that may betreated, prevented, and/or diagnosed with the compositions of theinvention (e.g., TR21 polynucleotides, polypeptides, and/or antagonists(e.g., anti-TR21 antibodies)) include, but are not limited to,autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis)(often characterized, e.g., by cell-mediated and humoral thyroidcytotoxicity), systemic lupus erhythematosus (often characterized, e.g.,by circulating and locally generated immune complexes), discoid lupus,Goodpasture's syndrome (often characterized, e.g., by anti-basementmembrane antibodies), Pemphigus (often characterized, e.g., by epidermalacantholytic antibodies), Receptor autoimmunities such as, for example,(a) Graves' Disease (often characterized, e.g., by TSH receptorantibodies), (b) Myasthenia Gravis (often characterized, e.g., byacetylcholine receptor antibodies), and (c) insulin resistance (oftencharacterized, e.g., by insulin receptor antibodies), autoimmunehemolytic anemia (often characterized, e.g., by phagocytosis ofantibody-sensitized RBCs), autoimmune thrombocytopenic purpura (oftencharacterized, e.g., by phagocytosis of antibody-sensitized platelets.

Additional autoimmune disorders (that are probable) that may be treated,prevented, and/or diagnosed with the compositions of the invention(e.g., TR21 polynucleotides, polypeptides, and/or antagonists (e.g.,anti-TR21 antibodies)) include, but are not limited to, rheumatoidarthritis (often characterized, e.g., by immune complexes in joints),scleroderma with anti-collagen antibodies (often characterized, e.g., bynucleolar and other nuclear antibodies), mixed connective tissue disease(often characterized, e.g., by antibodies to extractable nuclearantigens (e.g., ribonucleoprotein)), polymyositis/dermatomyositis (oftencharacterized, e.g., by nonhistone ANA), pernicious anemia (oftencharacterized, e.g., by antiparietal cell, microsomes, and intrinsicfactor antibodies), idiopathic Addison's disease (often characterized,e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility(often characterized, e.g., by antispermatozoal antibodies),glomerulonephritis (often characterized, e.g., by glomerular basementmembrane antibodies or immune complexes) such as primaryglomerulonephritis and IgA nephropathy, bullous pemphigoid (oftencharacterized, e.g., by IgG and complement in basement membrane),Sjogren's syndrome (often characterized, e.g., by multiple tissueantibodies, and/or a specific nonhistone ANA (SS-B)), diabetes mellitus(often characterized, e.g., by cell-mediated and humoral islet cellantibodies), and adrenergic drug resistance (including adrenergic drugresistance with asthma or cystic fibrosis) (often characterized, e.g.,by beta-adrenergic receptor antibodies).

Additional autoimmune disorders (that are possible) that may be treated,prevented, and/or diagnosed with the compositions of the invention(e.g., TR21 polynucleotides, polypeptides, and/or antagonists (e.g.,anti-TR21 antibodies)) include, but are not limited to, chronic activehepatitis (often characterized, e.g., by smooth muscle antibodies),primary biliary cirrhosis (often characterized, e.g., by mitchondrialantibodies), other endocrine gland failure (often characterized, e.g.,by specific tissue antibodies in some cases), vitiligo (oftencharacterized, e.g., by melanocyte antibodies), vasculitis (oftencharacterized, e.g., by Ig and complement in vessel walls and/or lowserum complement), post-MI (often characterized, e.g., by myocardialantibodies), cardiotomy syndrome (often characterized, e.g., bymyocardial antibodies), urticaria (often characterized, e.g., by IgG andIgM antibodies to IgE), atopic dermatitis (often characterized, e.g., byIgG and IgM antibodies to IgE), asthma (often characterized, e.g., byIgG and IgM antibodies to IgE), inflammatory myopathies, and many otherinflammatory, granulamatous, degenerative, and atrophic disorders.

In a preferred embodiment, the autoimmune diseases and disorders and/orconditions associated with the diseases and disorders recited above aretreated, prevented, and/or diagnosed using anti-TR21 antibodies.

In a specific preferred embodiment, multiple sclerosis is treated,prevented, and/or diagnosed anti-TR21 antibodies and/or other TR21antagonist of the invention.

In a specific preferred embodiment, rheumatoid arthritis is treated,prevented, and/or diagnosed anti-TR21 antibodies and/or other TR21antagonist of the invention.

In a specific preferred embodiment, lupus is treated, prevented, and/ordiagnosed using anti-TR21 antibodies and/or other TR21 antagonist of theinvention.

In a specific preferred embodiment, nephritis associated with lupus istreated, prevented, and/or diagnosed anti-TR21 antibodies and/or otherTR21 antagonist of the invention.

In a specific embodiment, TR21 polynucleotides or polypeptides, orantagonists thereof (e.g., anti-TR21 antibodies) are used to treat orprevent systemic lupus erythematosus and/or diseases, disorders orconditions associated therewith. Lupus-associated diseases, disorders,or conditions that may be treated or prevented with TR21 polynucleotidesor polypeptides, or antagonists of the invention, include, but are notlimited to, hematologic disorders (e.g., hemolytic anemia, leukopenia,lymphopenia, and thrombocytopenia), immunologic disorders (e.g.,anti-DNA antibodies, and anti-Sm antibodies), rashes, photosensitivity,oral ulcers, arthritis, fever, fatigue, weight loss, serositis (e.g.,pleuritus (pleuricy)), renal disorders (e.g., nephritis), neurologicaldisorders (e.g., seizures, peripheral neuropathy, CNS relateddisorders), gastroinstestinal disorders, Raynaud phenomenon, andpericarditis. In a preferred embodiment, the TR21 polynucleotides orpolypeptides, or antagonists thereof (e.g., anti-TR21 antibodies) areused to treat or prevent renal disorders associated with systemic lupuserythematosus. In a most preferred embodiment, TR21 polynucleotides orpolypeptides, or antagonists thereof (e.g., anti-TR21 antibodies) areused to treat or prevent nephritis associated with systemic lupuserythematosus.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by TR21 polynucleotides or polypeptides of the inventionand/or agonists and/or antagonists thereof. Moreover, these moleculescan be used to treat, prevent, and/or diagnose anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

TR21 polynucleotides or polypeptides of the invention and/or antagoniststhereof (e.g., antagonistic anti-TR21 antibodies), may also be used tomodulate blood clotting and to treat or prevent blood-clottingdisorders, such as, for example, antibody-mediated thrombosis (i.e.,antiphospholipid antibody syndrome (APS)). For example, TR21polynucleotides or polypeptides of the invention and/or antagoniststhereof, may inhibit the proliferation and differentiation of cellsinvolved in producing anticardiolipin antibodies. These compositions ofthe invention can be used to treat, prevent, and/or diagnose, thromboticrelated events including, but not limited to, stroke (and recurrentstroke), heart attack, deep vein thrombosis, pulmonary embolism,myocardial infarction, coronary artery disease (e.g., antibody-mediatedcoronary artery disease), thrombosis, graft reocclusion followingcardiovascular surgery (e.g., coronary arterial bypass grafts, recurrentfetal loss, and recurrent cardiovascular thromboembolic events.

TR21 polynucleotides or polypeptides of the invention and/or agonistsand/or antagonists thereof, may also be used to treat, prevent, and/ordiagnose organ rejection or graft-versus-host disease (GVHD) and/orconditions associated therewith. Organ rejection occurs by host immunecell destruction of the transplanted tissue through an immune response.Similarly, an immune response is also involved in GVHD, but, in thiscase, the foreign transplanted immune cells destroy the host tissues.The administration of TR21 polynucleotides or polypeptides of theinvention and/or agonists and/or antagonists thereof, that inhibits animmune response, particularly the proliferation, differentiation, orchemotaxis of T-cells, may be an effective therapy in preventing organrejection or GVHD.

Similarly, TR21 polynucleotides or polypeptides of the invention and/oragonists and/or antagonists thereof, may also be used to modulateinflammation. For example, TR21 polynucleotides or polypeptides of theinvention and/or agonists and/or antagonists thereof, may inhibit theproliferation and differentiation of cells involved in an inflammatoryresponse. These molecules can be used to treat, prevent, and/or diagnoseinflammatory conditions, both chronic and acute conditions, includingchronic prostatitis, granulomatous prostatitis and malacoplakia,inflammation associated with infection (e.g., septic shock, sepsis, orsystemic inflammatory response syndrome (SIRS)), ischemia-reperfusioninjury, endotoxin lethality, arthritis, complement-mediated hyperacuterejection, nephritis, cytokine or chemokine induced lung injury,inflammatory bowel disease, Crohn's disease, or due to overproduction ofcytokines (e.g., TNF or IL-1).

In a specific embodiment, anti-TR21 antibodies of the invention are usedto treat, prevent, modulate, detect, and/or diagnose inflammation.

In a specific embodiment, anti-TR21 antibodies of the invention are usedto treat, prevent, modulate, detect, and/or diagnose inflammatorydisorders.

In another specific embodiment, anti-TR21 antibodies of the inventionare used to treat, prevent, modulate, detect, and/or diagnose allergyand/or hypersensitivity.

Antibodies against TR21 may be employed to bind to and inhibit TR21activity to treat, prevent, and/or diagnose ARDS, by preventinginfiltration of neutrophils into the lung after injury. The agonists andantagonists of the instant may be employed in a composition with apharmaceutically acceptable carrier, e.g., as described hereinafter.

TR21 polynucleotides or polypeptides of the invention and/or agonistsand/or antagonists thereof (e.g., antagonistic anti-TR21 antibodies),may be used to treat, prevent, and/or diagnose diseases and disorders ofthe pulmonary system (e.g., bronchi such as, for example, sinopulmonaryand bronchial infections and conditions associated with such diseasesand disorders and other respiratory diseases and disorders. In specificembodiments, such diseases and disorders include, but are not limitedto, bronchial adenoma, bronchial asthma, pneumonia (such as, e.g.,bronchial pneumonia, bronchopneumonia, and tuberculousbronchopneumonia), chronic obstructive pulmonary disease (COPD),bronchial polyps, bronchiectasia (such as, e.g., bronchiectasia sicca,cylindrical bronchiectasis, and saccular bronchiectasis), bronchiolaradenocarcinoma, bronchiolar carcinoma, bronchiolitis (such as, e.g.,exudative bronchiolitis, bronchiolitis fibrosa obliterans, andproliferative bronchiolitis), bronchiolo-alveolar carcinoma, bronchiticasthma, bronchitis (such as, e.g., asthmatic bronchitis, Castellani'sbronchitis, chronic bronchitis, croupous bronchitis, fibrinousbronchitis, hemorrhagic bronchitis, infectious avian bronchitis,obliterative bronchitis, plastic bronchitis, pseudomembranousbronchitis, putrid bronchitis, and verminous bronchitis), bronchocentricgranulomatosis, bronchoedema, bronchoesophageal fistula, bronchogeniccarcinoma, bronchogenic cyst, broncholithiasis, bronchomalacia,bronchomycosis (such as, e.g., bronchopulmonary aspergillosis),bronchopulmonary spirochetosis, hemorrhagic bronchitis, bronchorrhea,bronchospasm, bronchostaxis, bronchostenosis, Biot's respiration,bronchial respiration, Kussmaul respiration, Kussmaul-Kien respiration,respiratory acidosis, respiratory alkalosis, respiratory distresssyndrome of the newborn, respiratory insufficiency, respiratoryscleroma, respiratory syncytial virus, and the like.

In a specific embodiment, TR21 polynucleotides or polypeptides of theinvention and/or agonists thereof (e.g., agonistic anti-TR21antibodies), are used to treat, prevent, and/or diagnose chronicobstructive pulmonary disease (COPD).

In another embodiment, TR21 polynucleotides or polypeptides of theinvention and/or agonists and/or antagonists thereof, are used to treat,prevent, and/or diagnose fibroses and conditions associated withfibroses, including, but not limited to, cystic fibrosis (including suchfibroses as cystic fibrosis of the pancreas, Clarke-Hadfield syndrome,fibrocystic disease of the pancreas, mucoviscidosis, and viscidosis),endomyocardial fibrosis, idiopathic retroperitoneal fibrosis,leptomeningeal fibrosis, mediastinal fibrosis, nodular subepidermalfibrosis, pericentral fibrosis, perimuscular fibrosis, pipestemfibrosis, replacement fibrosis, subadventitial fibrosis, and Symmers'clay pipestem fibrosis.

The TNF family ligands are known to be among the most pleiotropiccytokines, inducing a large number of cellular responses, includingcytotoxicity, anti-viral activity, immunoregulatory activities, and thetranscriptional regulation of several genes (D. V. Goeddel et al.,“Tumor Necrosis Factors: Gene Structure and Biological Activities,”Symp. Quant. Biol. 51:597- 609 (1986), Cold Spring Harbor; B. Beutlerand A. Cerami, Annu. Rev. Biochem. 57:505-518 (1988); L. J. Old, Sci.Am. 258:59-75 (1988); W. Fiers, FEBS Lett. 285:199-224 (1991). TheTNF-family ligands, including the TR21 ligands (e.g., Neutrokine-alpha),induce such various cellular responses by binding to TNF-familyreceptors, including TR21. TNF-ligand (e.g., Neutrokine-alpha)/TR21interactions may elicit a potent cellular response including anygenotypic, phenotypic, and/or morphologic change to the cell, cell line,tissue, tissue culture or patient. As indicated, such cellular responsesinclude not only normal physiological responses to TNF-family ligands,but such responses may lead to diseases associated with dysregulation ofthese physiological responses, such as, for example, diseases associatedwith increased apoptosis or the inhibition of apoptosis.Apoptosis-programmed cell death-is a physiological mechanism involved inthe deletion of peripheral B and/or T lymphocytes of the immune system,and its dysregulation can lead to a number of different pathogenicprocesses (J. C. Ameisen, AIDS 8:1197-1213 (1994); P. H. Krammer et al.,Curr. Opin. Immunol. 6:279-289 (1994)).

TR21 polynucleotides, polypeptides, agonists and/or antagonists (e.g.,agonistic antibodies) of the invention may be administered to a patient(e.g., mammal, preferably human) afflicted with any disease or disordermediated (directly or indirectly) by defective, or deficient levels of,TR21 or TR21 ligand (e.g., Neutrokine-alpha). Alternatively, a genetherapy approach may be applied to treat such diseases or disorders. Inone embodiment of the invention, TR21 polynucleotide sequences are usedto detect mutein TR21 genes, including defective genes. Mutein genes maybe identified in in vitro diagnostic assays, and by comparison of theTR21 nucleotide sequence disclosed herein with that of a TR21 geneobtained from a patient suspected of harboring a defect in this gene.Defective genes may be replaced with normal TR21-encoding genes usingtechniques known to one skilled in the art.

In another embodiment, the TR21 polypeptides, polynucleotides, agonistsand/or antagonists of the present invention are used as research toolsfor studying the phenotypic effects that result from inhibitingTR21/TR21 ligand interactions on various cell types. TR21 polypeptidesand antagonists (e.g. monoclonal antibodies to TR21) also may be used inin vitro assays for detecting TR21, TR21 ligands, or the interactionsthereof.

Diseases associated with increased cell survival, or the inhibition ofapoptosis that may be diagnosed, treated, or prevented with the TR21polynucleotides or polypeptides (including anti-TR21 antibodies) of theinvention, and agonists and antagonists thereof, include cancers (suchas follicular lymphomas, carcinomas with p53 mutations, andhormone-dependent tumors, including, but not limited to, colon cancer,cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomachcancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma,breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer);autoimmune disorders (such as systemic lupus erythematosus andimmune-related glomerulonephritis rheumatoid arthritis); viralinfections (such as herpes viruses, pox viruses and adenoviruses);inflammation; graft vs. host disease; acute graft rejection and chronicgraft rejection. Thus, in preferred embodiments TR21 polynucleotides orpolypeptides of the invention and/or agonists or antagonists thereof,are used to treat, prevent, and/or diagnose autoimmune diseases and/orinhibit the growth, progression, and/or metastasis of cancers,including, but not limited to, those cancers disclosed herein, such as,for example, lymphocytic leukemias (including, for example, MLL andchronic lymphocytic leukemia (CLL)) and follicular lymphomas. In anotherembodiment TR21 polynucleotides or polypeptides of the invention areused to activate, differentiate or proliferate cancerous cells or tissue(e.g., B cell lineage related cancers (e.g., CLL and MLL), lymphocyticleukemia, or lymphoma) and thereby render the cells more vulnerable tocancer therapy (e.g., chemotherapy or radiation therapy).

Moreover, in other embodiments, TR21 polynucleotides or polypeptides ofthe invention (including anti-TR21 antibodies) or agonists orantagonists thereof, may be used to inhibit the growth, progression,and/or metastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)), acute myelogenousleukemia, and chronic leukemias (e.g., chronic myelocytic (granulocytic)leukemia and chronic lymphocytic leukemia)), polycythemia vera,lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiplemyeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solidtumors including, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma. In specific embodiments, TR21 polynucleotides orpolypeptides (e.g., TR21 extracellular domain-Fc fusion proteins) of theinvention or agonists or antagonists thereof (e.g., anti-TR21antibodies) are used to diagnose, treat, or prevent acute myelogenousleukemia. In further specific embodiments, TR21 polypeptides or agonistsor antagonists (e.g., anti-TR21 antibodies) are used to diagnose, treat,or prevent chronic lymphocytic leukemia, multiple myeloma, non-Hodgkin'slymphoma, and/or Hodgkin's disease.

Diseases associated with increased apoptosis and that may be treated orprevented by the polynucleotides, polypeptides and/or agonists orantagonists of the invention include, but are not limited to, AIDS;neurodegenerative disorders (such as Alzheimer's disease, Parkinson'sdisease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellardegeneration and brain tumor or prior associated disease); autoimmunedisorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis); myelodysplastic syndromes(such as aplastic anemia), graft v. host disease, ischemic injury (suchas that caused by myocardial infarction, stroke and reperfusion injury),liver injury (such as hepatitis related liver injury,ischemia/reperfusion injury, cholestosis (bile duct injury) and livercancer); toxin-induced liver disease (such as that caused by alcohol),septic shock, cachexia and anorexia. In preferred embodiments, TR21polynucleotides, polypeptides and/or agonists are used to treat thediseases and disorders listed above.

In preferred embodiments, TR21 polypeptides of the invention and/oragonists or antagonists thereof (e.g., anti-TR21 antibodies) inhibit thegrowth of human histiocytic lymphoma U-937 cells in a dose-dependentmanner. In additional preferred embodiments, TR21 polypeptides of theinvention and/or agonists or antagonists thereof (e.g., anti-TR21antibodies) inhibit the growth of PC-3 cells, HT-29 cells, HeLa cells,MCF-7 cells, and A293 cells. In highly preferred embodiments, TR21polynucleotides or polypeptides of the invention and/or agonists orantagonists thereof (e.g., anti-TR21 antibodies) are used to inhibitgrowth, progression, and/or metastasis of prostate cancer, colon cancer,cervical carcinoma, and breast carcinoma.

Thus, in additional preferred embodiments, the present invention isdirected to a method for enhancing apoptosis induced by a TNF-familyligand, which involves administering to a cell which expresses TR21 aneffective amount of TR21 ligand (e.g., Neutrokine-alpha), or an agonistor antagonist of TR21, capable of increasing or decreasing TR21 mediatedsignaling. Preferably, TR21 mediated signaling is increased or decreasedto treat, prevent, and/or diagnose a disease wherein decreased apoptosisor decreased cytokine and adhesion molecule expression is exhibited. Anagonist or antagonist can include soluble forms of TR21 and monoclonalantibodies directed against the TR21 polypeptide.

Many of the pathologies associated with HIV are mediated by apoptosis,including HW-induced nephropathy and HIV encephalitis. Thus, inadditional preferred embodiments, TR21 polynucleotides, polypeptides,and/or TR21 agonists (e.g., anti-TR21 antibodies) or antagonists of theinvention are used to treat AIDS and pathologies associated with AIDS.

The state of immunodeficiency that defines AIDS is secondary to adecrease in the number and function of CD4⁺ T-lymphocytes. Recentreports estimate the daily loss of CD4⁺ T cells to be between 3.5×10⁷and 2×10⁹ cells (Wei et al., Nature 373:117-122 (1995)). One cause ofCD4⁺ T cell depletion in the setting of HIV infection is believed to beIIIV-induced apoptosis (see, for example, Meyaard et al., Science257:217-219, 1992; Groux et al., J Exp. Med., 175:331, 1992; and Oyaizuet al., in Cell Activation and Apoptosis in HIV Infection, Andrieu andLu, Eds., Plenum Press, New York, 1995, pp. 101-114). Indeed,HIV-induced apoptotic cell death has been demonstrated not only in vitrobut also, more importantly, in infected individuals (J. C. Ameisen, AIDS8:1197-1213 (1994); T. H. Finkel and N. K. Banda, Curr. Opin. Immunol.6:605-615(1995); C. A. Muro-Cacho et al., J Immunol. 154:5555-5566(1995)). Furthermore, apoptosis and CD4⁺ T-lymphocyte depletion istightly correlated in different animal models of AIDS (T. Brunner etal., Nature 373:441-444 (1995); M. L. Gougeon et al., AIDS Res. Hum.Retroviruses 9:553-563 (1993)) and, apoptosis is not observed in thoseanimal models in which viral replication does not result in AIDS. Id.Further data indicates that uninfected but primed or activated Tlymphocytes from HIV-infected individuals undergo apoptosis afterencountering the TNF-family ligand FasL. Using monocytic cell lines thatresult in death following HIV infection, it has been demonstrated thatinfection of U937 cells with HIV results in the de novo expression ofFasL and that FasL mediates HIV-induced apoptosis (A. D. Badley et al.,J Virol. 70:199-206 (1996)). Further, the TNF-family ligand wasdetectable in uninfected macrophages and its expression was upregulatedfollowing HIV infection resulting in selective killing of uninfected CD4T-lymphocytes. Id. Thus, by the invention, a method for treating HIV⁺individuals is provided which involves administering TR21 and/or TR21agonists (e.g., anti-TR21 antibodies) or antagonists of the presentinvention to reduce selective killing of CD4⁺ T-lymphocytes. Modes ofadministration and dosages are discussed in detail below.

Activated human T cells are induced to undergo programmed cell death(apoptosis) upon triggering through the CD3/T cell receptor complex, aprocess termed activated-induced cell death (AICD). AICD of CD4⁺ T cellsisolated from HIV-Infected asymptomatic individuals has been reported(Groux et al., supra). Thus, AICD may play a role in the depletion ofCD4⁺ T cells and the progression to AIDS in HIV-infected individuals.Thus, the present invention provides a method of inhibiting TNFligand-mediated T cell death in HIV patients, comprising administering aTR21 polypeptide of the invention (preferably, a soluble TR21polypeptide) to the patients. In one embodiment, the patient isasymptomatic when treatment with TR21 commences. If desired, prior totreatment, peripheral blood T cells may be extracted from an HIVpatient, and tested for susceptibility to TNF ligand-mediated cell deathby procedures known in the art. In one embodiment, a patient's blood orplasma is contacted with TR21 ex vivo. The TR21 may be bound to asuitable chromatography matrix by procedures known in the art. Thepatient's blood or plasma flows through a chromatography columncontaining TR21 bound to the matrix, before being returned to thepatient. The immobilized TR21 binds TNF ligand, thus removing TNF ligandprotein from the patient's blood.

In additional embodiments a TR21 polypeptide of the invention isadministered in combination with other inhibitors of T cell apoptosis.For example, Fas-mediated apoptosis and TRAIL-mediated apoptosis havealso has been implicated in loss of T cells in HIV individuals (See,e.g., Katsikis et al., J. Exp. Med. 181:2029-2036 (1995)). Thus, apatient susceptible to Fas ligand mediated and/or TRAIL mediated T celldeath may be treated with an agent that blocks Fas-ligand/Fas receptorinteractions and/or an agent that blocks TRAIL/TRAIL interactions.

Suitable agents for blocking binding of Fas-ligand to Fas that may beadministered with the TR21 polynucleotides or polypeptides of theinvention (including TR21 agonists (e.g., agonistic antibodies) and/orantagonists) include, but are not limited to, soluble Fas polypeptides;multimeric forms of soluble Fas polypeptides (e.g., dimers of sFas/Fc);anti-Fas antibodies that bind Fas without transducing the biologicalsignal that results in apoptosis; anti-Fas-ligand antibodies that blockbinding of Fas-ligand to Fas; and muteins of Fas-ligand that bind Fasbut do not transduce the biological signal that results in apoptosis.Preferably, the antibodies employed according to this method aremonoclonal antibodies. Examples of suitable agents for blockingFas-ligand/Fas interactions, including blocking anti-Fas monoclonalantibodies, are described in International application publicationnumber WO 95/10540, hereby incorporated by reference.

Suitable agents, which also block binding of TRAIL to a TRAIL receptorthat may be administered with the polynucleotides and/or polypeptides ofthe present invention include, but are not limited to, soluble TRAILreceptor polypeptides (e.g., a soluble form of OPG, DR4 (Internationalapplication publication number WO 98/32856); TR5 (Internationalapplication publication number WO 98/30693); and DR5 (Internationalapplication publication number WO 98/41629)); multimeric forms ofsoluble TRAIL receptor polypeptides; and TRAIL receptor antibodies thatbind the TRAIL receptor without transducing the biological signal thatresults in apoptosis, anti-TRAIL antibodies that block binding of TRAILto one or more TRAIL receptors, and muteins of TRAIL that bind TRAILreceptors but do not transduce the biological signal that results inapoptosis. Preferably, the antibodies employed according to this methodare monoclonal antibodies.

In a further aspect, the present invention is directed to a method forinhibiting apoptosis induced by a TNF-family ligand, which involvesadministering to a cell which expresses the TR21 an effective amount ofan agonist or antagonist capable of increasing or decreasing signalingmediated by the TR21. Preferably, TR21 mediated signaling is increasedor decreased to treat, prevent, and/or diagnose a disease whereinincreased apoptosis or NF-kappaB expression is exhibited. An agonist orantagonist can include soluble forms of TR21 and monoclonal antibodiesdirected against the TR21 polypeptide.

TR21 polypeptides or polynucleotides encoding TR21 and anti-TR21antibodies of the invention of the invention may be used to treatcardiovascular disorders, including peripheral artery disease, such aslimb ischemia.

Cardiovascular disorders include cardiovascular abnormalities, such asarterio-arterial fistula, arteriovenous fistula, cerebral arteriovenousmalformations, congenital heart defects, pulmonary atresia, and ScimitarSyndrome. Congenital heart defects include aortic coarctation, cortriatriatum, coronary vessel anomalies, crisscross heart, dextrocardia,patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex,hypoplastic left heart syndrome, levocardia, tetralogy of fallot,transposition of great vessels, double outlet right ventricle, tricuspidatresia, persistent truncus arteriosus, and heart septal defects, suchas aortopulmonary septal defect, endocardial cushion defects,Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septaldefects, and conditions characterized by clotting of small bloodvessels.

Cardiovascular disorders also include heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve diseases include aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,thrombotic microangiopathies (e.g., thrombotic thrombocytopenic purpura(TTP) and hemolytic-uremic syndrome (HUS)), and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans.

Cerebrovascular disorders include carotid artery diseases, cerebralamyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebralarteriosclerosis, cerebral arteriovenous malformation, cerebral arterydiseases, cerebral embolism and thrombosis, carotid artery thrombosis,sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epiduralhematoma, subdural hematoma, subarachnoid hemorrhage, cerebralinfarction, cerebral ischemia (including transient), subclavian stealsyndrome, periventricular leukomalacia, vascular headache, clusterheadache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterolembolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, andthromboembolisms. Thrombosis include coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includesaortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

The naturally occurring balance between endogenous stimulators andinhibitors of angiogenesis is one in which inhibitory influencespredominate. See e.g., Rastinejad et al., Cell 56:345-355 (1989). Inthose rare instances in which neovascularization occurs under normalphysiological conditions, such as wound healing, organ regeneration,embryonic development, and female reproductive processes, angiogenesisis stringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al., N Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442-447 (1987).

The present invention provides for treatment of diseases or disordersassociated with neovascularization by administration of the TR21polynucleotides and/or polypeptides of the invention (including TR21agonists (e.g., agonistic antibodies) and/or antagonists). Malignant andmetastatic conditions which can be treated with the polynucleotides andpolypeptides of the invention include, but are not limited to thosemalignancies, solid tumors, and cancers described herein and otherwiseknown in the art (for a review of such disorders, see Fishman et al.,Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).

Additionally, ocular disorders associated with neovascularization whichcan be treated with the TR21 polynucleotides and polypeptides of thepresent invention (including TR21 agonists and TR21 antagonists)include, but are not limited to: neovascular glaucoma, diabeticretinopathy, retinoblastoma, retrolental fibroplasia, uveitis,retinopathy of prematurity macular degeneration, corneal graftneovascularization, as well as other eye inflammatory diseases, oculartumors and diseases associated with choroidal or irisneovascularization. See, e.g., reviews by Waltman et al., Am. J.Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312(1978).

Additionally, disorders which can be treated with the TR21polynucleotides and polypeptides of the present invention (includingTR21 agonists and TR21 antagonists) include, but are not limited to,hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques,delayed wound healing, granulations, hemophilic joints, hypertrophicscars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma,scleroderma, trachoma, and vascular adhesions.

The polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof, can also be employed to inhibit theproliferation and differentiation of hematopoietic cells and thereforemay be employed to protect bone marrow stem cells from chemotherapeuticagents during chemotherapy. This antiproliferative effect may allowadministration of higher doses of chemotherapeutic agents and,therefore, more effective chemotherapeutic treatment.

The polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof, may also be employed for the expansion ofimmature hematopoietic progenitor cells, for example, granulocytes,macrophages or monocytes (e.g., C-kit+, Sca-1+), by temporarilypreventing their differentiation. These bone marrow cells may becultured in vitro. Thus, TR21 may be useful as a modulator ofhematopoietic stem cells in vitro for the purpose of bone marrowtransplantation and/or gene therapy. Since stem cells are rare and aremost useful for introducing genes into for gene therapy, TR21 can beused to isolate enriched populations of stem cells. Stem cells can beenriched by culturing cells in the presence of cytotoxins, such as 5-Fu,which kills rapidly dividing cells, where as the stem cells will beprotected by TR21. These stem cells can be returned to a bone marrowtransplant patient or can then be used for transfection of the desiredgene for gene therapy. In addition, TR21 can be injected into animalswhich results in the release of stem cells from the bone marrow of theanimal into the peripheral blood. These stem cells can be isolated forthe purpose of autologous bone marrow transplantation or manipulationfor gene therapy. After the patient has finished chemotherapy orradiation treatment, the isolated stem cells can be returned to thepatient.

In a specific embodiment, polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof may be used toincrease the concentration of blood cells in individuals in need of suchincrease (i.e., in hematopoietic therapy). Conditions that may beameliorated by administering the compositions of the invention include,but are not limited to, neutropenia, anemia, and thrombocytopenia.

In a specific embodiment, the polynucleotides and/or polypeptides of theinvention (and/or agonists or antagonists thereof) are used inerythropoietin therapy, which is directed toward supplementing theoxygen carrying capacity of blood. Polynucleotides and/or polypeptidesof the invention (and/or agonists or antagonists thereof) may be used totreat or prevent diseases or conditions in patients generally requiringblood transfusions, such as, for example, trauma victims, surgicalpatients, dialysis patients, and patients with a variety of bloodcomposition-affecting disorders, such as, for example, hemophilia,cystic fibrosis, pregnancy, menstrual disorders, early anemia ofprematurity, spinal cord injury, aging, various neoplastic diseasestates, and the like. Examples of patient conditions that requiresupplementation of the oxygen carrying capacity of blood and which arewithin the scope of this invention, include, but are not limited to:treatment of blood disorders characterized by low or defective red bloodcell production, anemia associated with chronic renal failure,stimulation of reticulocyte response, development of ferrokineticeffects (such as plasma iron turnover effects and marrow transit timeeffects), erythrocyte mass changes, stimulation of hemoglobin Csynthesis, and increasing levels of hematocrit in vertebrates. Theinvention also provides for treatment to enhance the oxygen-carryingcapacity of an individual, such as for example, an individualencountering hypoxic environmental conditions.

TR21 polynucleotides, polypeptides and/or agonists or antagonists mayalso be employed to regulate hematopoiesis, by regulating the activationand differentiation of various hematopoietic progenitor cells, forexample, to release mature leukocytes from the bone marrow followingchemotherapy, i.e., in stem cell mobilization. TR21 polynucleotides,polypeptides and/or agonists or antagonists may also be employed totreat sepsis.

TR21 polynucleotides, polypeptides and/or agonists or antagonists mayalso be employed to inhibit T-cell proliferation by the inhibition ofIL-2 biosynthesis for the treatment of T-cell mediated auto-immunediseases and lymphocytic leukemias (including, for example, chroniclymphocytic leukemia (CLL)).

TR21 polynucleotides, polypeptides and/or agonists or antagonists mayalso be employed to stimulate wound healing, both via the recruitment ofdebris clearing and connective tissue promoting inflammatory cells. Inthis same manner, TR21 polynucleotides, polypeptides and/or agonists orantagonists may also be employed to treat other fibrotic disorders,including liver cirrhosis, osteoarthritis and pulmonary fibrosis.

TR21 polynucleotides, polypeptides and/or agonists or antagonists mayalso be employed to enhance host defenses against resistant chronic andacute infections, for example, myobacterial infections via theattraction and activation of microbicidal leukocytes.

TR21 polynucleotides, polypeptides and/or agonists or antagonists alsoincreases the presence of eosinophils which have the distinctivefunction of killing the larvae of parasites that invade tissues, as inschistosomiasis, trichinosis and ascariasis.

Because TR21 belongs to the TNFR superfamily, the polypeptides shouldalso modulate angiogenesis. In addition, since TR21 inhibits immune cellfunctions, the polypeptides will have a wide range of anti-inflammatoryactivities. TR21 may be employed as an anti-neovascularizing agent totreat, prevent, and/or diagnose solid tumors by stimulating the invasionand activation of host defense cells, e.g., cytotoxic T cells andmacrophages and by inhibiting the angiogenesis of tumors. Those of skillin the art will recognize other non-cancer indications where bloodvessel proliferation is not wanted. They may also be employed to enhancehost defenses against resistant chronic and acute infections, forexample, myobacterial infections via the attraction and activation ofmicrobicidal leukocytes. TR21 may also be employed to inhibit T-cellproliferation by the inhibition of IL-2 biosynthesis for the treatmentof T-cell mediated auto-immune diseases and lymphocytic leukemias(including, for example, chronic lymphocytic leukemia (CLL)). TR21 mayalso be employed to stimulate wound healing, both via the recruitment ofdebris clearing and connective tissue promoting inflammatory cells. Inthis same manner, TR21 may also be employed to treat, prevent, and/ordiagnose other fibrotic disorders, including liver cirrhosis,osteoarthritis and pulmonary fibrosis. TR21 also increases the presenceof eosinophils that have the distinctive function of killing the larvaeof parasites that invade tissues, as in schistosomiasis, trichinosis andascariasis. It may also be employed to regulate hematopoiesis, byregulating the activation and differentiation of various hematopoieticprogenitor cells, for example, to release mature leukocytes from thebone marrow following chemotherapy, i.e., in stem cell mobilization.TR21 may also be employed to treat, prevent, and/or diagnose sepsis.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are useful in the diagnosis and treatment orprevention of a wide range of diseases and/or conditions. Such diseasesand conditions include, but are not limited to, cancer (e g , immunecell related cancers, breast cancer, prostate cancer, ovarian cancer,follicular lymphoma, cancer associated with mutation or alteration ofp53, brain tumor, bladder cancer, uterocervical cancer, colon cancer,colorectal cancer, non-small cell carcinoma of the lung, small cellcarcinoma of the lung, stomach cancer, etc.), lymphoproliferativedisorders (e.g., lymphadenopathy), microbial (e.g., viral, bacterial,etc.) infection (e.g., HIV-1 infection, HIV-2 infection, herpesvirusinfection (including, but not limited to, HSV-1, HSV-2, CMV, VZV, HHV-6,HHV-7, EBV), adenovirus infection, poxvirus infection, human papillomavirus infection, hepatitis infection (e.g., HAV, HBV, HCV, etc.),Helicobacter pylori infection, invasive Staphylococci, etc.), parasiticinfection, nephritis, bone disease (e.g., osteoporosis),atherosclerosis, pain, cardiovascular disorders (e.g.,neovascularization, hypovascularization or reduced circulation (e.g.,ischemic disease (e.g., myocardial infarction, stroke, etc.)), AIDS,allergy, inflammation, neurodegenerative disease (e.g., Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, pigmentaryretinitis, cerebellar degeneration, etc.), graft rejection (acute andchronic), graft vs. host disease, diseases due to osteomyelodysplasia(e.g., aplastic anemia, etc.), joint tissue destruction in rheumatism,liver disease (e.g., acute and chronic hepatitis, liver injury, andcirrhosis), autoimmune disease (e.g., multiple sclerosis, rheumatoidarthritis, systemic lupus erythematosus, immune complexglomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenicpurpura, Grave's disease, Hashimoto's thyroiditis, etc.), cardiomyopathy(e.g., dilated cardiomyopathy), diabetes, diabetic complications (e.g.,diabetic nephropathy, diabetic neuropathy, diabetic retinopathy),influenza, asthma, psoriasis, glomerulonephritis, septic shock, andulcerative colitis.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are useful in promoting angiogenesis, woundhealing (e.g., wounds, burns, and bone fractures). Polynucleotidesand/or polypeptides of the invention and/or agonists and/or antagoniststhereof are also useful as an adjuvant to enhance immune responsivenessto specific antigen, anti-viral immune responses.

More generally, polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof are useful in regulating(i.e., elevating or reducing) immune response. For example,polynucleotides and/or polypeptides of the invention may be useful inpreparation or recovery from surgery, trauma, radiation therapy,chemotherapy, and transplantation, or may be used to boost immuneresponse and/or recovery in the elderly and immunocompromisedindividuals. Alternatively, polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof are useful asimmunosuppressive agents, for example in the treatment or prevention ofautoimmune disorders. In specific embodiments, polynucleotides and/orpolypeptides of the invention are used to treat or prevent chronicinflammatory, allergic or autoimmune conditions, such as those describedherein or are otherwise known in the art.

In one aspect, the present invention is directed to a method forenhancing TR21 mediated signaling by a TNF-family ligand, which involvesadministering to a cell which expresses the TR21 polypeptide aneffective amount of TR21 ligand, analog or an agonist capable ofincreasing TR21 mediated signaling. Preferably, TR21 mediated signalingis increased to treat a disease wherein increased apoptosis, decreasedcytokine and adhesion molecule expression, or decreased cellproliferation is exhibited. An agonist can include soluble forms of TR21and monoclonal antibodies directed against the TR21 polypeptide.

In a further aspect, the present invention is directed to a method forinhibiting TR21 mediated signaling induced by a TNF-family ligand, whichinvolved administering to a cell which expresses the TR21 polypeptide aneffective amount of an antagonist capable of decreasing TR21 mediatedsignaling. Preferably, TR21 mediated signaling is decreased to treat adisease wherein decreased apoptosis or NF-kappaB expression, orincreased cell proliferation, is exhibited. An antagonist can includesoluble forms of TR21 and monoclonal antibodies directed against theTR21 polypeptide.

Preferably, treatment using TR21 polynucleotides or polypeptides, and/oragonists or antagonists of TR21 (e.g., anti-TR21 antibodies), couldeither be by administering an effective amount of TR21 polypeptide ofthe invention, or agonist or antagonist thereof, to the patient, or byremoving cells from the patient, supplying the cells with TR21polynucleotide, and returning the engineered cells to the patient (exvivo therapy). Moreover, as further discussed herein, the TR21polypeptide or polynucleotide can be used as an adjuvant in a vaccine toraise an immune response against infectious disease.

Formulations and Administration

The TR21 polypeptide composition (preferably containing anti-TR21antibody or a polypeptide which is a soluble form of the TR21extracellular domain) will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient (especially the side effects oftreatment with TR21 polypeptide alone), the site of delivery of the TR21polypeptide composition, the method of administration, the scheduling ofadministration, and other factors known to practitioners. The “effectiveamount” of TR21 polypeptide for purposes herein is thus determined bysuch considerations.

As a general proposition, the total pharmaceutically effective amount ofTR21 polypeptide administered parenterally per dose will be in the rangeof about 1 microgram/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day.

In another embodiment, the TR21 polypeptide of the invention isadministered to a human at a dose between 0.0001 and 0.045 mg/kg/day,preferably, at a dose between 0.0045 and 0.045 mg/kg/day, and morepreferably, at a dose of about 45 microgram/kg/day in humans; and at adose of about 3 mg/kg/day in mice.

If given continuously, the TR21 polypeptide is typically administered ata dose rate of about 1 microgram/kg/hour to about 50 micrograms/kg/hour,either by 1-4 injections per day or by continuous subcutaneousinfusions, for example, using a mini-pump. An intravenous bag solutionmay also be employed.

The length of treatment needed to observe changes and the intervalfollowing treatment for responses to occur appears to vary depending onthe desired effect.

In a specific embodiment, the total pharmaceutically effective amount ofTR21 polypeptide administered parenterally per dose will be in the rangeof about 0.1 microgram/kg/day to 45 micrograms/kg/day of patient bodyweight, although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.1 microgram/kg/day,and most preferably for humans between about 0.01 and 50micrograms/kg/day for the protein. TR21 polypeptides of the inventionmay be administered as a continuous infusion, multiple discreteinjections per day (e.g., three or more times daily, or twice daily),single injection per day, or as discreet injections given intermittently(e.g., twice daily, once daily, every other day, twice weekly, weekly,biweekly, monthly, bimonthly, and quarterly). If given continuously, theTR21 polypeptide is typically administered at a dose rate of about 0.001to 10 microgram/kg/ hour to about 50 micrograms/kg/hour, either by 1-4injections per day or by continuous subcutaneous infusions, for example,using a mini-pump.

Effective dosages of the compositions of the present invention to beadministered may be determined through procedures well known to those inthe art which address such parameters as biological half-life,bioavailability, and toxicity. Such determination is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

Bioexposure of an organism to TR21 polypeptide during therapy may alsoplay an important role in determining a therapeutically and/orpharmacologically effective dosing regime. Variations of dosing such asrepeated administrations of a relatively low dose of TR21 polypeptidefor a relatively long period of time may have an effect which istherapeutically and/or pharmacologically distinguishable from thatachieved with repeated administrations of a relatively high dose of TR21for a relatively short period of time.

Using the equivalent surface area dosage conversion factors supplied byFreireich, E. J., et al. (Cancer Chemotherapy Reports 50(4):219-44(1966)), one of ordinary skill in the art is able to convenientlyconvert data obtained from the use of TR21 in a given experimentalsystem into an accurate estimation of a pharmaceutically effectiveamount of TR21 polypeptide to be administered per dose in anotherexperimental system. Experimental data obtained through theadministration of TR21 in mice may converted through the conversionfactors supplied by Freireich, et al., to accurate estimates ofpharmaceutically effective doses of TR21 in rat, monkey, dog, and human.The following conversion table (Table III) is a summary of the dataprovided by Freireich, et al. Table III gives approximate factors forconverting doses expressed in terms of mg/kg from one species to anequivalent surface area dose expressed as mg/kg in another speciestabulated.

TABLE III Equivalent Surface Area Dosage Conversion Factors. TO MouseRat Monkey Dog Human FROM (20 g) (150 g) (3.5 kg) (8 kg) (60 kg) Mouse 1½ ¼ ⅙   1/12 Rat 2 1 ½ ¼ 1/7 Monkey 4 2 1 ⅗ ⅓ Dog 6 4 5/3 1 ½ Human 12 73 2 1

Thus, for example, using the conversion factors provided in Table III, adose of 50 mg/kg in the mouse converts to an appropriate dose of 12.5mg/kg in the monkey because (50 mg/kg)×(1/4)=12.5 mg/kg. As anadditional example, doses of 0.02, 0.08, 0.8, 2, and 8 mg/kg in themouse equate to effect doses of 1.667 micrograms/kg, 6.67 micrograms/kg,66.7 micrograms/kg, 166.7 micrograms/kg, and 0.667 mg/kg, respectively,in the human.

Pharmaceutical compositions containing TR21 polypeptides of theinvention may be administered orally, rectally, parenterally,subcutaneously, intracisternally, intravaginally, intraperitoneally,topically (as by powders, ointments, drops or transdermal patch),bucally, or as an oral or nasal spray (e.g., via inhalation of a vaporor powder). In one embodiment, “pharmaceutically acceptable carrier”means a non-toxic solid, semisolid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. In aspecific embodiment, “pharmaceutically acceptable” means approved by aregulatory agency of the federal or a state government or listed in theU.S. Pharmacopoeia or other generally recognized pharmacopoeia for usein animals, and more particularly humans. Nonlimiting examples ofsuitable pharmaceutical carriers according to this embodiment areprovided in “Remington's Pharmaceutical Sciences” by E. W. Martin, andinclude sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can be employed as liquid carriers, particularly forinjectable solutions. The composition, if desired, can also containminor amounts of wetting or emulsifying agents, or pH buffering agents.These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like.

The term “parenteral” as used herein refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal,intracistemal, subcutaneous and intraarticular injection and infusion.

In a preferred embodiment, TR21 compositions of the invention (includingpolypeptides, polynucleotides, and antibodies, and agonists and/orantagonists thereof) are administered subcutaneously.

In another preferred embodiment, TR21 compositions of the invention(including polypeptides, polynucleotides, and antibodies, and agonistsand/or antagonists thereof) are administered intravenously.

For parenteral administration, in one embodiment, the TR21 polypeptideis formulated generally by mixing it at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the TR21polypeptide uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,sucrose, or dextrins; chelating agents such as EDTA; sugar alcohols suchas mannitol or sorbitol; counterions such as sodium; preservatives, suchas cresol, phenol, chlorobutanol, benzyl alcohol and parabens, and/ornonionic surfactants such as polysorbates, poloxamers, or PEG.

The TR21 polypeptide is typically formulated in such vehicles at aconcentration of about 0.001 mg/ml to 100 mg/ml, or 0.1 mg/ml to 100mg/ml, preferably 1-10 mg/ml or 1-10 mg/ml, at a pH of about 3 to 10, or3 to 8, more preferably 5-8, most preferably 6-7. It will be understoodthat the use of certain of the foregoing excipients, carriers, orstabilizers will result in the formation of TR21 polypeptide salts.

TR21 polypeptide to be used for therapeutic administration must besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeutic TR21polypeptide compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

TR21 polypeptide ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous TR21 polypeptide solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized TR21 polypeptide using bacteriostaticWater-for-Injection.

Alternatively, TR21 polypeptide is stored in single dose containers inlyophilized form. The infusion selection is reconstituted using asterile carrier for injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally, associatedwith such container(s) is a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptides of the present invention may be employed inconjunction with other therapeutic compounds.

Pharmaceutical compositions of the present invention for parenteralinjection can comprise pharmaceutically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like.

In addition to soluble TR21 polypeptides, TR21 polypeptides containingthe transmembrane region can also be used when appropriately solubilizedby including detergents, such as CHAPS or NP-40, with buffer.

TR21 compositions of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releasecompositions include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556(1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J.Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech.12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Sustained-release compositions also include liposomally entrappedcompositions of the invention (see generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317 -327 and 353-365 (1989)). Liposomes containing TR21polypeptide may be prepared by methods known per se: DE 3,218,121;Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwanget al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal a polypeptide therapy.

In another embodiment sustained release compositions of the inventioninclude crystal formulations known in the art.

In yet an additional embodiment, the compositions of the invention aredelivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

The compositions of the invention may be administered alone or incombination with other adjuvants. Adjuvants that may be administeredwith the compositions of the invention include, but are not limited to,alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, compositionsof the invention are administered in combination with alum. In anotherspecific embodiment, compositions of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe compositions of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the compositions of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diphtheria, hepatitis A, hepatitis B, haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme disease, rotavirus, cholera,yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoidfever, and pertussis, and/or PNEUMOVAX-23™. Combinations may beadministered either concomitantly, e.g., as an admixture, separately butsimultaneously or concurrently; or sequentially. This includespresentations in which the combined agents are administered together asa therapeutic mixture, and also procedures in which the combined agentsare administered separately but simultaneously, e.g., as throughseparate intravenous lines into the same individual. Administration “incombination” further includes the separate administration of one of thecompounds or agents given first, followed by the second.

In a specific embodiment, compositions of the invention may beadministered to patients as vaccine adjuvants. In a further specificembodiment, compositions of the invention may be administered as vaccineadjuvants to patients suffering from an immune-deficiency. In a furtherspecific embodiment, compositions of the invention may be administeredas vaccine adjuvants to patients suffering from HIV.

In a specific embodiment, compositions of the invention may be used toincrease or enhance antigen-specific antibody responses to standard andexperimental vaccines. In a specific embodiment, compositions of theinvention may be used to enhance seroconversion in patients treated withstandard and experimental vaccines. In another specific embodiment,compositions of the invention may be used to increase the repertoire ofantibodies recognizing unique epitopes in response to standard andexperimental vaccination.

In a preferred embodiment, TR21 polypeptides of the invention (includingTR21 fragments and variants, and anti-TR21 antibodies) increase orenhance antigen-specific antibody responses to standard and experimentalvaccines by regulating binding of the soluble form of a TR21 ligand(e.g., Neutrokine-alpha, Neutroline-alpha SV and/or one or moreheteromultimeric polypeptide complexes comprising one or moreNeutrokine-alpha and/or Neutrokine-alpha SV polypeptides), to TR21and/or another Neutrokine-alpha receptor (e.g., TACI and/or BCMA).

In a preferred embodiment, TR21 polypeptides of the invention (includingTR21 fragments and variants, and anti-TR21 antibodies) increase orenhance seroconversion in patients treated with standard andexperimental vaccines by regulating binding of the soluble form of aTR21 ligand (e.g., Neutrokine-alpha, Neutroline-alpha SV and/or one ormore heteromultimeric polypeptide complexes comprising one or moreNeutrokine-alpha and/or Neutrokine-alpha SV polypeptides), to TR21and/or another Neutrokine alpha receptor (e.g., TACI and/or BCMA).

In a preferred embodiment, TR21 polypeptides of the invention (includingTR21 fragments and variants, and anti-TR21 antibodies) increase orenhance the repertoire of antibodies recognizing unique epitopes inresponse to standard and experimental vaccination by regulating bindingof the soluble form of a TR21 ligand (e.g., Neutrokine-alpha,Neutroline-alpha SV and/or one or more heteromultimeric polypeptidecomplexes comprising one or more Neutrokine-alpha and/orNeutrokine-alpha SV polypeptides), to TR21 and/or another Neutrokinealpha receptor (e.g., TACI and/or BCMA).

In another specific embodiment, compositions of the invention are usedin combination with PNEUMOVAX-23™ to treat, prevent, and/or diagnoseinfection and/or any disease, disorder, and/or condition associatedtherewith. In one embodiment, compositions of the invention are used incombination with PNEUMOVAX-23™ to treat, prevent, and/or diagnose anyGram-positive bacterial infection and/or any disease, disorder, and/orcondition associated therewith. In another embodiment, compositions ofthe invention are used in combination with PNEUMOVAX-23™ to treat,prevent, and/or diagnose infection and/or any disease, disorder, and/orcondition associated with one or more members of the genus Enterococcusand/or the genus Streptococcus. In another embodiment, compositions ofthe invention are used in any combination with PNEUMOVAX-23™ to treat,prevent, and/or diagnose infection and/or any disease, disorder, and/orcondition associated with one or more members of the Group Bstreptococci. In another embodiment, compositions of the invention areused in combination with PNEUMOVAX-23™ to treat, prevent, and/ordiagnose infection and/or any disease, disorder, and/or conditionassociated with Streptococcus pneumoniae.

The compositions of the invention may be administered alone or incombination with other therapeutic agents, including but not limited to,chemotherapeutic agents, antibiotics, antivirals, steroidal andnon-steroidal anti-inflammatories, conventional immunotherapeutic agentsand cytokines. Combinations may be administered either concomitantly,e.g., as an admixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the compositions of the invention are administered incombination with other members of the TNF family. TNF, TNF-related orTNF-like molecules that may be administered with the compositions of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), TRAIL, AIM-II (International Publication No. WO 97/34911),APRIL (International Publication Number WO 97/33902; J. Exp. Med.188(6):1185-1190) (1998)), endokine-alpha (International Publication No.WO 98/07880), Neutrokine-alpha and/or Neutrokine-alpha SV (InternationalApplication Publication No. WO 98/18921), OPG, OX40, and nerve growthfactor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2(International Publication No. WO 96/34095), DR3 (InternationalPublication No. WO 97/33904), DR4 (International Publication No. WO98/32856), TR5 (International Publication No. WO 98/30693), TR6(International Publication No. WO 98/30694), TR7 (InternationalPublication No. WO 98/41629), TRANK, TR9 (International Publication No.WO 98/56892), 312C2 (International Publication No. WO 98/06842), TR12,TACI (See, e.g., U.S. Pat. No. 5,969,102; and von Bulow et al., Science278:138-141 (1997)), CD154, CD70, and CD153.

In a preferred embodiment, the compositions of the invention areadministered in combination with CD40 ligand (CD40L), a soluble form ofCD40L (e.g., AVREND™), biologically active fragments, variants, orderivatives of CD40L, anti-CD40L antibodies (e.g., agonistic orantagonistic antibodies), and/or anti-CD40 antibodies (e.g., agonisticor antagonistic antibodies).

In a preferred embodiment, the compositions of the invention areadministered in combination with TACI (See e.g., U.S. Pat. No.5,969,102; and von Bulow et al., Science 278:138-141 (1997)), a solubleform of TACI, biologically active fragments, variants, or derivatives ofTACI (e.g., TACI-Fc), and/or anti-TACI antibodies (e.g., agonistic orantagonistic antibodies).

In a preferred embodiment, the compositions of the invention areadministered in combination with Neutrokine-alpha and/orNeutrokine-alpha SV (International Publication No. WO 98/18921), asoluble form of Neutrokine alpha and/or Neutrokine-alpha SV,biologically active fragments, variants, or derivatives ofNeutrokine-alpha and/or Neutrokine-alpha SV, and/oranti-Neutrokine-alpha and/or anti-Neutrokine-alpha SV antibodies (e.g.,agonistic or antagonistic antibodies).

In a preferred embodiment, the compositions of the invention areadministered in combination with APRIL (International Publication NumberWO 97/33902; J. Exp. Med. 188(6):1185-1190 (1998)), a soluble form ofAPRIL, biologically active fragments, variants, or derivatives of APRIL,and/or anti-APRIL antibodies (e.g., agonistic or antagonisticantibodies).

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-angiogenic agent(s).Anti-angiogenic agents that may be administered with the compositions ofthe invention include, but are not limited to, Angiostatin (Entremed,Rockville, MD), Troponin-1 (Boston Life Sciences, Boston, Mass.),anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel(Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, TissueInhibitor of Metalloproteinase-2, VEGI, Plasminogen ActivatorInhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of thelighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium, and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate and orthovanadate complexes such as, forexample, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude, but are not limited to, platelet factor 4; protamine sulphate;sulphated chitin derivatives (prepared from queen crab shells), (Murataet al., Cancer Res. 51:22-26, 1991); Sulphated PolysaccharidePeptidoglycan Complex (SP-PG) (the function of this compound may beenhanced by the presence of steroids such as estrogen, and tamoxifencitrate); Staurosporine; modulators of matrix metabolism, including forexample, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline,Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;(Takeuchi et al., Agents Actions 36:312-316, 1992); andmetalloproteinase inhibitors such as BB94.

Additional anti-angiogenic factors that may also be utilized within thecontext of the present invention include Thalidomide, (CELGENE™, Warren,N.J.); Angiostatic steroid; AGM-1470 (H. Brem and J. Folkman, J Pediatr.Surg. 28:445-51 (1993)); an integrin alpha v beta 3 antagonist (C.Storgard et al., J Clin. Invest. 103:47-54 (1999));carboxyaminolmidazole; Carboxyamidotriazole (CAI) (National CancerInstitute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXiGENE™, Boston,Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.);TNP-470, (TAP PHARMACEUTICALS™, Deerfield, Ill.); ZD-0101 ASTRAZENECA™(London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251(PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin;Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide(Somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat(AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (NOLVADEX™);Tazarotene; Tetrathiomolybdate; XELODA™ (Capecitabine); and5-Fluorouracil.

Anti-angiogenic agents that may be administered in combination with thecompounds of the invention may work through a variety of mechanismsincluding, but not limited to, inhibiting proteolysis of theextracellular matrix, blocking the function of endothelialcell-extracellular matrix adhesion molecules, by antagonizing thefunction of angiogenesis inducers such as growth factors, and inhibitingintegrin receptors expressed on proliferating endothelial cells.Examples of anti-angiogenic inhibitors that interfere with extracellularmatrix proteolysis and which may be administered in combination with thecompositions of the invention include, but are not limited to, AG-3340(AGOURON™, La Jolla, Calif.), BAY-12-9566 (BAYER™, West Haven, Conn.),BMS-275291 (BRISTOL MYERS SQUIBB™, Princeton, N.J.), CGS-27032A(NOVARTIS™, East Hanover, N.J.), MARIMASTAT™ (BRITISH BIOTECH™, Oxford,UK), and METASTAT™ (AETERNA™, St-Foy, Quebec). Examples ofanti-angiogenic inhibitors that act by blocking the function ofendothelial cell-extracellular matrix adhesion molecules and which maybe administered in combination with the compositions of the inventioninclude, but are not limited to, EMD-121974 (MERCK™ KcgaA Darmstadt,Germany) and VITAXIN™ (IXSYSTM, La Jolla, Calif./MEDIMMUNE™,Gaithersburg, Md.). Examples of anti-angiogenic agents that act bydirectly antagonizing or inhibiting angiogenesis inducers and which maybe administered in combination with the compositions of the inventioninclude, but are not limited to, ANGIOZYME™ (RIBOZYME™, Boulder, Colo.),Anti-VEGF antibody (GENENTECH™, S. San Francisco, Calif.),PTK-787/ZK-225846 (NOVARTIS™, Basel, Switzerland), SU-101 (SUGEN™, S.San Francisco, Calif.), SU-5416 (SUGEN™/PHARMACIA™ Upjohn, Bridgewater,N.J.), and SU-6668 (SUGEN™). Other anti-angiogenic agents act toindirectly inhibit angiogenesis. Examples of indirect inhibitors ofangiogenesis which may be administered in combination with thecompositions of the invention include, but are not limited to, IM-862(CYTRAN™, Kirkland, Wash.), Interferon-alpha, IL-12 (ROCHE™, Nutley,N.J.), and Pentosan polysulfate (Georgetown University, Washington,D.C.).

In particular embodiments, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of an autoimmune disease,such as for example, an autoimmune disease described herein.

In a particular embodiment, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of arthritis. In a moreparticular embodiment, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of rheumatoid arthritis orconditions associated therewith.

In a particular embodiment, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of arthritis. In a moreparticular embodiment, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of systemic lupuserythematosus or conditions associated therewith.

In another embodiment, compositions of the invention are administered incombination with an anticoagulant. Anticoagulants that may beadministered with the compositions of the invention include, but are notlimited to, heparin, warfarin, and aspirin. In a specific embodiment,compositions of the invention are administered in combination withheparin and/or warfarin. In another specific embodiment, compositions ofthe invention are administered in combination with warfarin. In anotherspecific embodiment, compositions of the invention are administered incombination with warfarin and aspirin. In another specific embodiment,compositions of the invention are administered in combination withheparin. In another specific embodiment, compositions of the inventionare administered in combination with heparin and aspirin.

In another embodiment, compositions of the invention are administered incombination with an agent that suppresses the production ofanticardiolipin antibodies. In specific embodiments, the polynucleotidesof the invention are administered in combination with an agent thatblocks and/or reduces the ability of anticardiolipin antibodies to bindphospholipid-binding plasma protein beta 2-glycoprotein I (b2GPI).

In another embodiment, therapeutic or pharmaceutical compositions of theinvention are administered to an animal to treat, prevent or ameliorateischemia and arteriosclerosis. Examples of such disorders include, butare not limited to, reperfusion damage (e.g., in the heart and/or brain)and cardiac hypertrophy.

Therapeutic or pharmaceutical compositions of the invention may also beadministered to modulate blood clotting and to treat or preventblood-clotting disorders, such as, for example, antibody-mediatedthrombosis (i.e., antiphospholipid antibody syndrome (APS)). Forexample, therapeutic or pharmaceutical compositions of the invention mayinhibit the proliferation and differentiation of cells involved inproducing anticardiolipin antibodies. These compositions of theinvention can be used to treat, prevent, ameliorate, diagnose, and/orprognose thrombotic related events including, but not limited to, stroke(and recurrent stroke), heart attack, deep vein thrombosis, pulmonaryembolism, myocardial infarction, coronary artery disease (e.g.,antibody-mediated coronary artery disease), thrombosis, graftreocclusion following cardiovascular surgery (e.g., coronary arterialbypass grafts, recurrent fetal loss, and recurrent cardiovascularthromboembolic events.

In certain embodiments, compositions of the invention are administeredin combination with antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the compositionsof the invention, include, but are not limited to, RETROVIR™(zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC),ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith compositions of the invention to treat, prevent, and/or diagnoseAIDS and/or to treat, prevent, and/or diagnose HIV infection.

Additional NRTIs include LODENOSINE™ (F-ddA; an acid-stable adenosineNRTI; Triangle/Abbott; COVIRACIL™ (emtricitabine/FTC; structurallyrelated to lamivudine (3TC) but with 3- to 10-fold greater activity invitro; Triangle/Abbott); dOTC (BCH-10652, also structurally related tolamivudine but retains activity against a substantial proportion oflamivudine-resistant isolates; Biochem Pharma); Adefovir (refusedapproval for anti-HIV therapy by FDA; Gilead Sciences); PREVEON®(Adefovir Dipivoxil, the active prodrug of adefovir; its active form isPMEA-pp); TENOFOVIR™ (bis-POC PMPA, a PMPA prodrug; Gilead); DAPD/DXG(active metabolite of DAPD; Triangle/Abbott); D-D4FC (related to 3TC,with activity against AZT/3TC-resistant virus); GW420867X (GlaxoWellcome); ZIAGEN™ (abacavir/159U89; Glaxo Wellcome Inc.); CS-87(3′azido-2′, 3′-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl(SATE)-bearing prodrug forms of β-L-FD4C and β-L-FddC (WO 98/17281).

Additional NNRTIs include COACTINON™ (Emivirine/MKC-442, potent NNRTI ofthe HEPT class; Triangle/Abbott); CAPRAVIRINE™ (AG-1549/S-1153, a nextgeneration NNRTI with activity against viruses containing the K103Nmutation; AGOURON™); PNU-142721 (has 20- to 50-fold greater activitythan its predecessor delavirdine and is active against K103N mutants;PHARMACIA™ & Upjohn); DPC-961 and DPC-963 (second-generation derivativesof efavirenz, designed to be active against viruses with the K103Nmutation; DuPont); GW-420867X (has 25-fold greater activity than HBY097and is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A(naturally occurring agent from the latex tree; active against virusescontaining either or both the Y181C and K103N mutations); and Propolis(WO 99/49830).

Additional protease inhibitors include LOPINAVIR™ (ABT378/r; AbbottLaboratories); BMS-232632 (an azapeptide; Bristol-Myers Squibb);TIPRANAVIR™ (PNU-140690, a non-peptic dihydropyrone; PHARIVIACIA™ &Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS232632 (an azapeptide; Bristol-Myers Squibb); L-756,423 (an indinaviranalog; Merck); DMP-450 (a cyclic urea compound; Avid & DuPont); AG-1776(a peptidomimetic with in vitro activity against proteaseinhibitor-resistant viruses; AGOURON™); VX-175/GW-433908 (phosphateprodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755 (Ciba); andAGENERASE™ (amprenavir; Glaxo Wellcome Inc.).

Additional antiretroviral agents include fusion inhibitors/gp41 binders.Fusion inhibitors/gp41 binders include T-20 (a peptide from residues643-678 of the HIV gp41 transmembrane protein ectodomain which binds togp41 in its resting state and prevents transformation to the fusogenicstate; Trimeris) and T-1249 (a second-generation fusion inhibitor;Trimeris).

Additional antiretroviral agents include fusion inhibitors/chemokinereceptor antagonists. Fusion inhibitors/chemokine receptor antagonistsinclude CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and itsanalogs, and ALX40-4C (a cationic peptide), T22 (an 18 amino acidpeptide; Trimeris) and the T22 analogs T134 and T140; CCRS antagonistssuch as RANTES (9-68), AOP-RANTES , NNY-RANTES, and TAK-779; andCCR5/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Alsoincluded are CCR2B, CCR3, and CCR6 antagonists. Chemokine receptoragonists such as RANTES, SDF-1, MIP-1α, MIP-1β, etc., may also inhibitfusion.

Additional antiretroviral agents include integrase inhibitors. Integraseinhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (adicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and relatedanthraquinones; ZINTEVIR™ (AR 177, an oligonucleotide that probably actsat cell surface rather than being a true integrase inhibitor; Arondex);and naphthols such as those disclosed in WO 98/50347.

Additional antiretroviral agents include hydroxyurea-like compounds suchas BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst);ribonucleotide reductase inhibitors such as DIDOX™ (Molecules forHealth); inosine monophosphate dehydrogenase (IMPDH) inhibitors such asVX-497 (Vertex); and myvopholic acids such as CellCept (mycophenolatemofetil; ROCHE™)

Additional antiretroviral agents include inhibitors of viral integrase,inhibitors of viral genome nuclear translocation such as arylenebis(methylketone) compounds; inhibitors of HIV entry such as AOP-RANTES,NNY-RANTES, RANTES-IgG fusion protein, soluble complexes of RANTES andglycosaminoglycans (GAG), and AMD-3100; nucleocapsid zinc fingerinhibitors such as dithiane compounds; targets of HIV Tat and Rev; andpharmacoenhancers such as ABT-378.

Other antiretroviral therapies and adjunct therapies include cytokinesand lymphokines such as MIP-1α, MIP-1β, SDF-1α, IL-2, PROLEUKIN™(aldesleukin/L2-7001; CHIRON™), IL-4, IL-10, IL-12, and IL-13;interferons such as IFN-α2a; antagonists of TNFs, NFKB, GM-CSF, M-CSF,and IL-10; agents that modulate immune activation such as cyclosporinand prednisone; vaccines such as Remune™ (HIV Immunogen), APL 400-003(Apollon), recombinant gp120 and fragments, bivalent (B/E) recombinantenvelope glycoprotein, rgp120CM235, MN rgp120, SF-2 rgp120,gp120/soluble CD4 complex, Delta JR-FL protein, branched syntheticpeptide derived from discontinuous gp120 C3/C4 domain, fusion-competentimmunogens, and Gag, Pol, Nef, and Tat vaccines; gene-based therapiessuch as genetic suppressor elements (GSEs; WO 98/54366), and intrakines(genetically modified CC chemokines targeted to the ER to block surfaceexpression of newly synthesized CCR5 (Yang et al., PNAS 94:11567-72(1997); Chen et al., Nat. Med. 3:1110-16 (1997)); antibodies such as theanti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9,PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA-T4,the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b, 48d,447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies, anti-TNF-α,antibodies, and monoclonal antibody 33A; aryl hydrocarbon (AII) receptoragonists and antagonists such as TCDD, 3,3′, 4,4′,5-pentachlorobiphenyl, 3,3′,4,4′-tetrachlorobiphenyl, andα-naphthoflavone (WO 98/30213); and antioxidants such asγ-L-glutamyl-L-cysteine ethyl ester (γ-GCE; WO 99/56764).

In other embodiments, compositions of the invention may be administeredin combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe compositions of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, compositions of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat, prevent, and/or diagnose anopportunistic Pneumocystis carnii pneumonia infection. In anotherspecific embodiment, compositions of the invention are used in anycombination with ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/orETHAMBUTOL™ to prophylactically treat, prevent, and/or diagnose anopportunistic Mycobacterium avium complex infection. In another specificembodiment, compositions of the invention are used in any combinationwith RIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ toprophylactically treat, prevent, and/or diagnose an opportunisticMycobacterium tuberculosis infection. In another specific embodiment,compositions of the invention are used in any combination withGANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylactically treat,prevent, and/or diagnose an opportunistic cytomegalovirus infection. Inanother specific embodiment, compositions of the invention are used inany combination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™to prophylactically treat, prevent, and/or diagnose an opportunisticfungal infection. In another specific embodiment, compositions of theinvention are used in any combination with ACYCLOVIR™ and/orFAMCICOLVIR™ to prophylactically treat, prevent, and/or diagnose anopportunistic herpes simplex virus type I and/or type II infection. Inanother specific embodiment, compositions of the invention are used inany combination with PYRIMETHAMINE™ and/or LEUCOVORIN™ toprophylactically treat, prevent, and/or diagnose an opportunisticToxoplasma gondii infection. In another specific embodiment,compositions of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat, prevent, and/ordiagnose an opportunistic bacterial infection.

In a further embodiment, the compositions of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the compositions of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, amoxicillin, aminoglycosides, beta-lactam(glycopeptide), beta-lactamases, Clindamycin, chloramphenicol,cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin,fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim,trimethoprim-sulfamethoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs cyclophosphamide, cyclophosphamide IV, methylprednisolone,prednisolone, azathioprine, FK-506, 15-deoxyspergualin, and otherimmunosuppressive agents that act by suppressing the function ofresponding T cells. Other immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, prednisolone, methotrexate,thalidomide, methoxsalen, rapamycin, leflunomide, mizoribine(BREDININ™), brequinar, deoxyspergualin, and azaspirane (SKF 105685).

In specific embodiments, compositions of the invention are administeredin combination with immunosuppressants. Immunosuppressants preparationsthat may be administered with the compositions of the invention include,but are not limited to, ORTHOCLONE™ (OKT3), (muromonab-CD3),SANDIMMUNE™, NEORAL™, SANGDYA™ (cyclosporine), PROGRAF® (FK506,tacrolimus), CELLCEPT® (mycophenolate motefil, of which the activemetabolite is mycophenolic acid), IMURAN™ (azathioprine),corticosteroids, adrenocortical steroids such as DELTASONE™ (prednisone)and HYDELTRASOL™ (prednisolone), FOLEX™ and MEXATE™ (methotrexate),OXSORALEN-ULTRA™ (methoxsalen) and RAPAMUNE™ (sirolimus). In a specificembodiment, immunosuppressants may be used to prevent rejection of organor bone marrow transplantation.

In a preferred embodiment, the compositions of the invention areadministered in combination with steroid therapy. Steroids that may beadministered in combination with the compositions of the invention,include, but are not limited to, oral corticosteroids, prednisone, andmethylprednisolone (e.g., IV methylprednisolone). In a specificembodiment, compositions of the invention are administered incombination with prednisone. In a further specific embodiment, thecompositions of the invention are administered in combination withprednisone and an immunosuppressive agent. Immunosuppressive agents thatmay be administered with the compositions of the invention andprednisone are those described herein, and include, but are not limitedto, azathioprine, cyclophosphamide, and cyclophosphamide IV. In anotherspecific embodiment, compositions of the invention are administered incombination with methylprednisolone. In a further specific embodiment,the compositions of the invention are administered in combination withmethylprednisolone and an immunosuppressive agent. Immunosuppressiveagents that may be administered with the compositions of the inventionand methylprednisolone are those described herein, and include, but arenot limited to, azathioprine, cyclophosphamide, and cyclophosphamide IV.

In a preferred embodiment, the compositions of the invention areadministered in combination with an antimalarial. Antimalarials that maybe administered with the compositions of the invention include, but arenot limited to, hydroxychloroquine, chloroquine, and/or quinacrine.

In a preferred embodiment, the compositions of the invention areadministered in combination with an NSAID.

In a nonexclusive embodiment, the compositions of the invention areadministered in combination with one, two, three, four, five, ten, ormore of the following drugs: NRD-101 (HOECHST MARION ROUSSEL™)diclofenac (Dimethaid), oxaprozin potassium (MONSANTO™), mecasermin(CHIRON™), T-614 (TOYAMA™), pemetrexed disodium (ELI LILLY™), atreleuton(ABBOTT™), valdecoxib (MONSANTO™), eltenac (Byk Gulden), CAMPATH™,AGM-1470 (TAKEDA™), CDP-571 (CELLTECH CHIROSCIENCE™), CM-101 (CarboMed),ML-3000 (Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix), IL-1Ragene therapy (VALENTIS™), JTE-522 (JAPAN TOBACCO™), paclitaxel(ANGIOTECH™), DW-166HC (Dong Wha), darbufelone mesylate(WARNER-LAMBERT™), soluble TNF receptor 1 (SYNERGEN™; AMGEN™), IPR-6001(Institute for Pharmaceutical Research), trocade (HOFFMAN-LA ROCHE™),EF-5 (SCOTIA PHARMACEUTICALS™), BIIL-284 (BOEHRINGER INGELHEIM™),BIIF-1149 (BOEHRINGER INGELHEIM™), LEUKOVAX™ (INFLAMMATICS™), MK-663(MERCK™), ST-1482 (Sigma-Tau), and butixocort propionate(WARNER-LAMBERT™).

In one embodiment, the compositions of the invention are administered incombination with one or more of the following drugs: infliximab (alsoknown as REMICADE™ Centocor, Inc.), Trocade (ROCHE™, RO-32-3555),Leflunomide (also known as ARAVA™ from HOECHST MARION ROUSSEL™),KINERET™ (an IL-1 Receptor antagonist also known as Anakinra from Amgen,Inc.), SCID-469 (p38 kinase inhibitor from Scios, Inc), and/or ASLERA™(prasterone, dehydroepiandrosterone, GL701) from Genelabs TechnologiesInc.

In a preferred embodiment, the compositions of the invention areadministered in combination with one, two, three, four, five or more ofthe following drugs: methotrexate, sulfasalazine, sodium aurothiomalate,auranofin, cyclosporine, penicillamine, azathioprine, an antimalarialdrug (e.g., as described herein), cyclophosphamide, chlorambucil, gold,ENBREL™ (Etanercept), anti-TNF antibody, LJP 394 (La JollaPharmaceutical Company, San Diego, Calif.) and prednisolone.

In a more preferred embodiment, the compositions of the invention areadministered in combination with an antimalarial, methotrexate, anti-TNFantibody, ENBREL™ and/or suflasalazine. In one embodiment, thecompositions of the invention are administered in combination withmethotrexate. In another embodiment, the compositions of the inventionare administered in combination with anti-TNF antibody. In anotherembodiment, the compositions of the invention are administered incombination with methotrexate and anti-TNF antibody. In anotherembodiment, the compositions of the invention are administered incombination with suflasalazine. In another specific embodiment, thecompositions of the invention are administered in combination withmethotrexate, anti-TNF antibody, and suflasalazine. In anotherembodiment, the compositions of the invention are administered incombination ENBREL™. In another embodiment, the compositions of theinvention are administered in combination with ENBREL™ and methotrexate.In another embodiment, the compositions of the invention areadministered in combination with ENBREL™, methotrexate andsuflasalazine. In another embodiment, the compositions of the inventionare administered in combination with ENBREL™, methotrexate andsuflasalazine. In other embodiments, one or more antimalarials arecombined with one of the above-recited combinations. In a specificembodiment, the compositions of the invention are administered incombination with an antimalarial (e.g., hydroxychloroquine), ENBREL™,methotrexate and suflasalazine. In another specific embodiment, thecompositions of the invention are administered in combination with anantimalarial (e.g., hydroxychloroquine), sulfasalazine, anti-TNFantibody, and methotrexate.

In an additional embodiment, compositions of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the compositions of the invention include, but notlimited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, andGAMIMUNE™. In a specific embodiment, compositions of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the compositions of the invention areadministered in combination with CD40 ligand (CD40L), a soluble form ofCD40L (e.g., AVREND™), biologically active fragments, variants, orderivatives of CD40L, anti-CD40L antibodies (e.g., agonistic orantagonistic antibodies), and/or anti-CD40 antibodies (e.g., agonisticor antagonistic antibodies).

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the compositionsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compositions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the compositions of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,methoxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, compositions of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or any combination of the components of CHOP. In oneembodiment, the compositions of the invention are administered incombination with anti-CD20 antibodies, human monoclonal anti-CD20antibodies. In another embodiment, the compositions of the invention areadministered in combination with anti-CD20 antibodies and CHOP, oranti-CD20 antibodies and any combination of one or more of thecomponents of CHOP, particularly cyclophosphamide and/or prednisone. Ina specific embodiment, compositions of the invention are administered incombination with Rituximab. In a further embodiment, compositions of theinvention are administered with Rituximab and CHOP, or Rituximab and anycombination of one or more of the components of CHOP, particularlycyclophosphamide and/or prednisone. In a specific embodiment,compositions of the invention are administered in combination withtositumomab (anti-CD20 antibody from Coulter Pharmaceuticals, SanFrancisco, Calif.). In a further embodiment, compositions of theinvention are administered with tositumomab and CHOP, or tositumomab andany combination of one or more of the components of CHOP, particularlycyclophosphamide and/or prednisone. Tositumomab may optionally beassociated with ¹³¹I. The anti-CD20 antibodies may optionally beassociated with radioisotopes, toxins or cytotoxic prodrugs.

In another specific embodiment, the compositions of the invention areadministered in combination Zevalin™. In a further embodiment,compositions of the invention are administered with Zevalin™ and CHOP,or Zevalin™ and any combination of one or more of the components ofCHOP, particularly cyclophosphamide and/or prednisone. Zevalin™ may beassociated with one or more radioisotopes. Particularly preferredisotopes are ⁹⁰Y and ¹¹¹In.

In an additional embodiment, the compositions of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, GM-CSF, G-CSF, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12,IL13, IL15, anti-CD40, CD40L, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha,and TNF-beta. In another embodiment, compositions of the invention maybe administered with any interleukin, including, but not limited to,IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, IL-21, and IL-22. In preferred embodiments, the compositions ofthe invention are administered in combination with IL4 and IL10.

In one embodiment, the compositions of the invention are administered incombination with one or more chemokines. In specific embodiments, thecompositions of the invention are administered in combination with anα(C×C) chemokine selected from the group consisting of gamma-interferoninducible protein-10 (γIP-10), interleukin-8 (IL-8), platelet factor-4(PF4), neutrophil activating protein (NAP-2), GRO-α, GRO-β, GRO-γ,neutrophil-activating peptide (ENA-78), granulocyte chemoattractantprotein-2 (GCP-2), and stromal cell-derived factor-1 (SDF-1, or pre-Bcell stimulatory factor (PBSF)); and/or a β(CC) chemokine selected fromthe group consisting of: RANTES (regulated on activation, normal Texpressed and secreted), macrophage inflammatory protein-1 alpha(MIP-1α), macrophage inflammatory protein-1 beta (MIP-1β), monocytechemotactic protein-1 (MCP-1), monocyte chemotactic protein-2 (MCP-2),monocyte chemotactic protein-3 (MCP-3), monocyte chemotactic protein-4(MCP-4) macrophage inflammatory protein-1 gamma (MIP-1γ), macrophageinflammatory protein-3 alpha (MIP-3α), macrophage inflammatory protein-3beta (MIP-3β), macrophage inflammatory protein-4 (MIP-4/DC-CK-1/PARC),eotaxin, Exodus, and I-309; and/or the γ(C) chemokine, lymphotactin.

In another embodiment, the compositions of the invention areadministered with chemokine beta-8, chemokine beta-1, and/or macrophageinflammatory protein-4. In a preferred embodiment, the compositions ofthe invention are administered with chemokine beta-8.

In an additional embodiment, the compositions of the invention areadministered in combination with an IL-4 antagonist. IL-4 antagoniststhat may be administered with the compositions of the invention include,but are not limited to: soluble IL-4 receptor polypeptides, multimericforms of soluble IL-4 receptor polypeptides; anti-IL-4 receptorantibodies that bind the IL-4 receptor without transducing thebiological signal elicited by IL-4, anti-IL4 antibodies that blockbinding of IL-4 to one or more IL-4 receptors, and muteins of IL-4 thatbind IL-4 receptors but do not transduce the biological signal elicitedby IL-4. Preferably, the antibodies employed according to this methodare monoclonal antibodies (including antibody fragments, such as, forexample, those described herein).

In an additional embodiment, the compositions of the invention areadministered in combination with an IL-13 antagonist. IL-13 antagoniststhat may be administered with the compositions of the invention include,but are not limited to: soluble IL-13 receptor polypeptides, multimericforms of soluble IL-13 receptor polypeptides; anti-IL-13 receptorantibodies that bind the IL-13 receptor without transducing thebiological signal elicited by IL-13, anti-IL-13 antibodies that blockbinding of IL-13 to one or more IL-13 receptors, and muteins of IL-13that bind IL-13 receptors but do not transduce the biological signalelicited by IL-13. Preferably, the antibodies employed according to thismethod are monoclonal antibodies (including antibody fragments, such as,for example, those described herein).

The invention also encompasses combining the polynucleotides and/orpolypeptides of the invention (and/or agonists or antagonists thereof)with other proposed or conventional hematopoietic therapies. Thus, forexample, the polynucleotides and/or polypeptides of the invention(and/or agonists or antagonists thereof) can be combined with compoundsthat singly exhibit erythropoietic stimulatory effects, such aserythropoietin, testosterone, progenitor cell stimulators, insulin-likegrowth factor, prostaglandins, serotonin, cyclic AMP, prolactin, andtriiodothyzonine. Also encompassed are combinations of the compositionsof the invention with compounds generally used to treat aplastic anemia,such as, for example, methenolene, stanozolol, and nandrolone; to treatiron-deficiency anemia, such as, for example, iron preparations; totreat malignant anemia, such as, for example, vitamin B₁₂ and/or folicacid; and to treat hemolytic anemia, such as, for example,adrenocortical steroids, e.g., corticoids. See e.g., Resegotti et al.,Panminerva Medica, 23:243-248 (1981); Kurtz, FEBS Letters, 14a:105-108(1982); McGonigle et al., Kidney Int., 25:437-444 (1984); andPavlovic-Kantera, Expt. Hematol., 8(supp. 8) 283-291 (1980), thecontents of each of which are hereby incorporated by reference in theirentireties.

Compounds that enhance the effects of or synergize with erythropoietinare also useful as adjuvants herein, and include but are not limited to,adrenergic agonists, thyroid hormones, androgens, hepatic erythropoieticfactors, erythrotropins, and erythrogenins, See for e.g., Dunn, “CurrentConcepts in Erythropoiesis”, John Wiley and Sons (Chichester, England,1983); Kalmani, Kidney Int., 22:383-391 (1982); Shahidi, New Eng. J.Med., 289:72-80 (1973); Urabe et al., J. Exp. Med., 149:1314-1325(1979); Billat et al., Expt. Hematol., 10:133-140 (1982); Naughton etal., Acta Haemat, 69:171-179 (1983); Cognote et al. in abstract 364,Proceedings 7th Intl. Cong. of Endocrinology (Quebec City, Quebec, Jul.1-7, 1984); and Rothman et al., 1982, J. Surg. Oncol., 20:105-108(1982). Methods for stimulating hematopoiesis comprise administering ahematopoietically effective amount (i.e., an amount which effects theformation of blood cells) of a pharmaceutical composition containingpolynucleotides and/or polypeptides of the invention (and/or agonists orantagonists thereof) to a patient. The polynucleotides and/orpolypeptides of the invention and/or agonists or antagonists thereof isadministered to the patient by any suitable technique, including but notlimited to, parenteral, sublingual, topical, intrapulmonary andintranasal, and those techniques further discussed herein. Thepharmaceutical composition optionally contains one or more members ofthe group consisting of erythropoietin, testosterone, progenitor cellstimulators, insulin-like growth factor, prostaglandins, serotonin,cyclic AMP, prolactin, triiodothyzonine, methenolene, stanozolol, andnandrolone, iron preparations, vitamin B₁₂, folic acid and/oradrenocortical steroids.

In an additional embodiment, the compositions of the invention areadministered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with thecompositions of the invention include, but are not limited to, LEUKINE™(SARGRAMOSTIM™) and NEUPOGEN™ (FILGRASTIM™).

In an additional embodiment, the compositions of the invention areadministered in combination with fibroblast growth factors. Fibroblastgrowth factors that may be administered with the compositions of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

Additionally, the compositions of the invention may be administeredalone or in combination with other therapeutic regimens, including butnot limited to, radiation therapy. Such combinatorial therapy may beadministered sequentially and/or concomitantly.

Agonists and Antagonists—Assays and Molecules

The invention also provides a method of screening compounds to identifythose compounds which enhance or block the action of TR21 polypeptide oncells, such as its interaction with TR21 binding molecules such asligand molecules. An agonist is a compound which increases the naturalbiological functions of TR21 or which functions in a manner similar toTR21 while antagonists decrease or eliminate such functions.

In another embodiment, the invention provides a method for identifying aligand protein or other ligand-binding protein which binds specificallyto TR21 polypeptide. For example, a cellular compartment, such as amembrane or a preparation thereof, may be prepared from a cell thatexpresses a molecule that binds Neutrokine-alpha. The preparation isincubated with labeled Neutrokine-alpha and complexes ofNeutrokine-alpha bound to TR21 or other binding protein are isolated andcharacterized according to routine methods known in the art.Alternatively, the TR21 ligand polypeptide may be bound to a solidsupport so that binding molecules solubilized from cells are bound tothe column and then eluted and characterized according to routinemethods.

In the assay of the invention for agonists or antagonists, a cellularcompartment, such as a membrane or a preparation thereof, may beprepared from a cell that expresses a molecule that binds TR21 such as amolecule of a signaling or regulatory pathway modulated by TR21. Thepreparation is incubated with labeled TR21 in the absence or thepresence of a candidate molecule which may be a TR21 agonist orantagonist. The ability of the candidate molecule to bind the bindingmolecule is reflected in decreased binding of the labeled ligand.Molecules which bind gratuitously, i.e., without inducing the effects ofTR21 on binding the TR21 binding molecule, are most likely to be goodantagonists. Molecules that bind well and elicit effects that are thesame as or closely related to TR21 are agonists.

By “agonist” is intended naturally occurring and synthetic compoundscapable of enhancing or potentiating TR21 mediated signaling. By“antagonist” is intended naturally occurring and synthetic compoundscapable of inhibiting apoptosis. Whether any candidate “agonist” or“antagonist” of the present invention can enhance or inhibit TR21mediated signaling can be determined using art-known TNF-familyligand/receptor cellular response assays, including those described inmore detail below.

One such screening procedure involves the use of melanophores which aretransfected to express the receptor of the present invention. Such ascreening technique is described in PCT WO 92/01810. Such an assay maybe employed, for example, for screening for a compound which inhibits(or enhances) activation of the receptor polypeptide of the presentinvention by contacting the melanophore cells which encode the receptorwith both a TNF-family ligand and the candidate antagonist (or agonist).Inhibition or enhancement of the signal generated by the ligandindicates that the compound is an antagonist or agonist of theligand/receptor signaling pathway.

Other screening techniques include the use of cells which express thereceptor (for example, transfected CHO cells) in a system which measuresextracellular pH changes caused by receptor activation. For example,compounds may be contacted with a cell which expresses the receptorpolypeptide of the present invention and a second messenger response,e.g., signal transduction or pH changes, may be measured to determinewhether the potential compound activates or inhibits the receptor.

Another such screening technique involves introducing RNA encoding thereceptor into Xenopus oocytes to transiently express the receptor. Thereceptor oocytes may then be contacted with the receptor ligand and acompound to be screened, followed by detection of inhibition oractivation of a calcium signal in the case of screening for compoundswhich are thought to inhibit activation of the receptor.

Another screening technique well known in the art involves expressing incells a construct wherein the receptor is linked to a phospholipase C orD. Exemplary cells include endothelial cells, smooth muscle cells,embryonic kidney cells, etc. The screening may be accomplished ashereinabove described by detecting activation of the receptor orinhibition of activation of the receptor from the phospholipase signal.

Another method involves screening for compounds which inhibit activationof the receptor polypeptide of the present invention antagonists bydetermining inhibition of binding of labeled ligand to cells which havethe receptor on the surface thereof. Such a method involves transfectinga eukaryotic cell with DNA encoding the receptor such that the cellexpresses the receptor on its surface and contacting the cell with acompound in the presence of a labeled form of a known ligand. The ligandcan be labeled, e.g., by radioactivity. The amount of labeled ligandbound to the receptors is measured, e.g., by measuring radioactivity ofthe receptors. If the compound binds to the receptor as determined by areduction of labeled ligand which binds to the receptors, the binding oflabeled ligand to the receptor is inhibited.

Further screening assays for agonists and antagonists of the presentinvention are described in L. A.Tartaglia and D. V. Goeddel, J. Biol.Chem. 267:4304-4307(1992).

Thus, in a further aspect, a screening method is provided fordetermining whether a candidate agonist or antagonist is capable ofenhancing or inhibiting a cellular response to a TNF-family ligand. Themethod involves contacting cells which express the TR21 polypeptide witha candidate compound and a TNF-family ligand, assaying a cellularresponse, and comparing the cellular response to a standard cellularresponse, the standard being assayed when contact is made with theligand in absence of the candidate compound, whereby an increasedcellular response over the standard indicates that the candidatecompound is an agonist of the ligand/receptor signaling pathway and adecreased cellular response compared to the standard indicates that thecandidate compound is an antagonist of the ligand/receptor signalingpathway. By “assaying a cellular response” is intended qualitatively orquantitatively measuring a cellular response to a candidate compoundand/or a TNF-family ligand (e.g., determining or estimating an increaseor decrease in B and/or T cell proliferation or tritiated thymidinelabeling). By the invention, a cell expressing the TR21 polypeptide canbe contacted with either an endogenous or exogenously administeredTNF-family ligand.

TR21-like effects of potential agonists and antagonists may by measured,for instance, by determining activity of a second messenger systemfollowing interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that of TR21or molecules that elicit the same effects as TR21. Second messengersystems that may be useful in this regard include but are not limited toAMP guanylate cyclase, ion channel or phosphoinositide hydrolysis secondmessenger systems.

Another example of an assay for TR21 antagonists is a competitive assaythat combines TR21 and a potential antagonist with membrane-bound ligandmolecules or recombinant TR21 ligand molecules under appropriateconditions for a competitive inhibition assay. TR21 can be labeled, suchas by radioactivity, such that the number of TR21 molecules bound to aligand molecule can be determined accurately to assess the effectivenessof the potential antagonist.

Agonists according to the present invention include naturally occurringand synthetic compounds such as, for example, the CD40 ligand, neutralamino acids, zinc, estrogen, androgens, viral genes (such as AdenovirusEIB, Baculovirus p35 and IAP, Cowpox virus crmA, Epstein-Barr virusBHRF1, LMP-1, African swine fever virus LMW5-HL, and Herpesvirus y134.5), calpain inhibitors, cysteine protease inhibitors, and tumorpromoters (such as PMA, Phenobarbital, and α-Hexachlorocyclohexane).

Antagonist according to the present invention include naturallyoccurring and synthetic compounds such as, for example, TNF familyligand peptide fragments, transforming growth factor, neurotransmitters(such as glutamate, dopamine, N-methyl-D-aspartate), tumor suppressors(p53), cytolytic T cells and antimetabolites. Preferred agonists includechemotherapeutic drugs such as, for example, cisplatin, doxorubicin,bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate andvincristine. Others include ethanol and -amyloid peptide. (Science267:1457-1458 (1995)). Further preferred agonists include TR21polypeptides of the invention, polyclonal and monoclonal antibodiesraised against the TR21 polypeptide, or a fragment thereof. Such agonistantibodies raised against a TNF-family receptor are disclosed in L. A.Tartaglia et al., Proc. Natl. Acad. Sci. USA 88:9292-9296 (1991); and L.A. Tartaglia and D. V. Goeddel, J. Biol. Chem. 267:4304-4307(1992). See,also, PCT Application WO 94/09137.

Potential antagonists include small organic molecules, peptides,polypeptides (e.g., IL-13), and antibodies that bind to a polypeptide ofthe invention and thereby inhibit or extinguish its activity. Potentialantagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a ligand molecule, withoutinducing TR21 induced activities, thereby preventing the action of TR21by excluding TR21 from binding.

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through antisense DNAor RNA or through triple-helix formation. Antisense techniques arediscussed, for example, in Okano, J. Neurochem. 56: 560 (1991);“Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Antisense technology can be used tocontrol gene expression through antisense DNA or RNA, or throughtriple-helix formation. Antisense techniques are discussed for example,in Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Triple helix formation is discussed in, for instance Lee et al.,Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456(1988); and Dervan et al., Science 251: 1360 (1991). The methods arebased on binding of a polynucleotide to a complementary DNA or RNA. Forexample, the 5′ coding portion of a polynucleotide that encodes theextracellular domain of the polypeptide of the present invention may beused to design an antisense RNA oligonucleotide of from about 10 to 40base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription therebypreventing transcription and the production of TR21. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into TR21 polypeptide. The oligonucleotides describedabove can also be delivered to cells such that the antisense RNA or DNAmay be expressed in vivo to inhibit production of TR21.

In one embodiment, the TR21 antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the TR21 antisense nucleic acid. Such avector can remain episomal or become chromosomally integrated, as longas it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others know inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding TR21, or fragments thereof, can beby any promoter known in the art to act in vertebrate, preferably humancells. Such promoters can be inducible or constitutive. Such promotersinclude, but are not limited to, the SV40 early promoter region (Bemoistand Chambon, Nature 29:304-310 (1981)), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell22:787-797 (1980)), the herpes thymidine promoter (Wagner et al., Proc.Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981)), the regulatory sequencesof the metallothionein gene (Brinster, et al., Nature 296:39-42 (1982)),etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a TR21 gene.However, absolute complementarity, although preferred, is not required.A sequence “complementary to at least a portion of an RNA,” referred toherein, means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case of doublestranded TR21 antisense nucleic acids, a single strand of the duplex DNAmay thus be tested, or triplex formation may be assayed. The ability tohybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid. Generally, the larger thehybridizing nucleic acid, the more base mismatches with a TR21 RNA itmay contain and still form a stable duplex (or triplex as the case maybe). One skilled in the art can ascertain a tolerable degree of mismatchby use of standard procedures to determine the melting point of thehybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333-335. Thus, oligonucleotides complementary to either the 5′- or3′- non- translated, non-coding regions of TR21 shown in FIG. 1,respectively, could be used in an antisense approach to inhibittranslation of endogenous TR21 mRNA. Oligonucleotides complementary tothe 5′ untranslated region of the mRNA should include the complement ofthe AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but could beused in accordance with the invention. Whether designed to hybridize tothe 5′-, 3′- or coding region of TR21 mRNA, antisense nucleic acidsshould be at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Inspecific aspects the oligonucleotide is at least 10 nucleotides, atleast 17 nucleotides, at least 25 nucleotides or at least 50nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556; Lemaitre et al., Proc. Natl. Acad. Sci. 84:648-652(1987); PCT Publication No. WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., BioTechniques 6:958-976 (1988)) or intercalatingagents. (See, e.g., Zon, Pharm. Res. 5:539-549 (1988)). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is analpha-anomeric oligonucleotide. An alpha-anomeric oligonucleotide formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual beta-units, the strands run parallel to each other(Gautier et al., Nucl. Acids Res. 15:6625-6641 (1987)). Theoligonucleotide is a 2-0-methylribonucleotide (Inoue et al., Nucl. AcidsRes. 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al.,FEBS Lett. 215:327-330 (1997)).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (Nucl. Acids Res. 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A.85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the TR21 coding regionsequence could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published October 4, 1990; Sarver et al, Science 247:1222-1225(1990). While ribozymes that cleave mRNA at site-specific recognitionsequence can be used to destroy TR21 mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).There are numerous potential hammerhead ribozyme cleavage sites withinthe nucleotide sequence TR21 (FIG. 1). Preferably, the ribozyme isengineered so that the cleavage recognition site is located near the 5′end of the TR21 mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express TR21 invivo. DNA constructs encoding the ribozyme may be introduced into thecell in the same manner as described above for the introduction ofantisense encoding DNA. A preferred method of delivery involves using aDNA construct “encoding” the ribozyme under the control of a strongconstitutive promoter, such as, for example, pol III or pol II promoter,so that transfected cells will produce sufficient quantities of theribozyme to destroy endogenous TR21 messages and inhibit translation.Since ribozymes unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

Endogenous gene expression can also be reduced by inactivating or“knocking out” the TR21 gene and/or its promoter using targetedhomologous recombination. (E.g., see Smithies et al., Nature 317:230-234(1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart. The contents of each of the documents recited in this paragraph isherein incorporated by reference in its entirety.

In other embodiments, antagonists according to the present inventioninclude soluble forms of TR21 (e.g., fragments of TR21 shown in FIG. 1(SEQ ID NO:2) that include one or more copies of the cysteine rich motiffrom the extracellular domain of TR21). Such soluble forms of the TR21,which may be naturally occurring or synthetic, antagonize TR21 mediatedsignaling by competing with native TR21 for binding to TR21 ligands(e.g., Neutrokine-alpha (See, U.S. Application Ser. No. 60/188,208)),and/or by forming a multimer that may or may not be capable of bindingthe receptor, but which is incapable of inducing signal transduction.Preferably, these antagonists inhibit TR21 mediated stimulation oflymphocyte (e.g., B-cell) proliferation, differentiation, and/oractivation. Antagonists of the present invention also include antibodiesspecific for TNFR-family receptors and TR21-Fc fusion proteins.

By a “TNF-family ligand” is intended naturally occurring, recombinant,and synthetic ligands that are capable of binding to a member of the TNFreceptor family and inducing and/or blocking the ligand/receptorsignaling pathway. Members of the TNF ligand family include, but are notlimited to, TNF-alpha, lymphotoxin-alpha (LT-alpha, also known asTNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), FasL,CD40L, (TNF-gamma (International Publication No. WO 96/14328), AIM-I(International Publication No. WO 97/33899), AIM-II (InternationalPublication No. WO 97/34911), APRIL (International Publication Number WO97/33902; J. Exp. Med. 188(6):1185-1190) (1998)), endokine-alpha(International Publication No. WO 98/07880), Neutrokine-alpha(International Publication No. WO 98/18921), CD27L, CD30L, 4-1BBL,OX40L, CD27, CD30, 4-1BB, OX40, and nerve growth factor (NGF). Inspecific embodiments, the TNF-family ligand is Neutrokine-alpha, orfragments or variants thereof. In other specific embodiments, theTNF-family ligand is APRIL or fragments or variants thereof.

Antagonists of the present invention also include antibodies specificfor TNF-family ligands or the TR21 polypeptides of the invention.Antibodies according to the present invention may be prepared by any ofa variety of standard methods using TR21 immunogens of the presentinvention. As indicated, such TR21 immunogens include the complete TR21polypeptides depicted in FIG. 1 (SEQ ID NO:2) and TR21 polypeptidefragments comprising, for example, the cysteine rich domain,extracellular domain, transmembrane domain, and/or intracellular domain,or any combination thereof.

Polyclonal and monoclonal antibody agonists or antagonists according tothe present invention can be raised according to the methods disclosedherein and/or known in the art, such as, for example, those methodsdescribed in Tartaglia and Goeddel, J. Biol. Chem.267(7):4304-4307(1992)); Tartaglia et al., Cell 73:213-216 (1993)), andPCT Application WO 94/09137 and are preferably specific to (i.e., binduniquely to polypeptides of the invention having the amino acid sequenceof SEQ ID NO:2.

In a preferred method, antibodies according to the present invention aremAbs. Such mAbs can be prepared using hybridoma technology (Kohler andMilstein, Nature 256:495-497 (1975) and U.S. Pat. No. 4,376,110; Harlowet al., Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988; Monoclonal Antibodies andHybridomas: A New Dimension in Biological Analyses, Plenum Press, NewYork, N.Y., 1980; Campbell, “Monoclonal Antibody Technology,” In:Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13(Burdon et al., eds.), Elsevier, Amsterdam (1984)).

Antagonists according to the present invention include soluble forms ofTR21, i.e., TR21 fragments that include one or more of the cysteine richmotif from the extracellular region of the full-length receptor (orvariants thereof). Such soluble forms of the receptor, which may benaturally occurring or synthetic, antagonize TR21 mediated signaling bycompeting with the cell surface TR21 for binding to TNF-family ligands(e.g., Neutrokine-alpha). Thus, soluble forms of the receptor thatinclude one or more copies of the cysteine-rich motif of TR21 are novelcytokines capable of inhibiting TR21 mediated signaling induced byTNF-family ligands. These soluble forms are preferably expressed asdimers or trimers, since these have been shown to be superior tomonomeric forms of soluble receptor as antagonists, e.g., IgGFc-TNFreceptor family fusions. Other such cytokines are known in the art andinclude Fas B (a soluble form of the mouse Fas receptor) that actsphysiologically to limit apoptosis induced by Fas ligand (D. P. Hughesand I. N. Crispe, J. Exp. Med. 182:1395-1401 (1995)).

The techniques of gene shuffling, motif shuffling, exon shuffling,and/or codon-shuffling (collectively referred to as “DNA shuffling”) maybe employed to modulate the activities of TR21 thereby effectivelygenerating agonists and antagonists of TR21. See generally,International Publication No. WO 99/29902, U.S. Pat. Nos. 5,605,793,5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten et al., Curr.Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol.16(2):76-82 (1998); Hansson et al., J. Mol. Biol. 287:265-76 (1999); andLorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of thesepatents and publications are hereby incorporated by reference). In oneembodiment, alteration of TR21 polynucleotides and correspondingpolypeptides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments into a desired TR21 molecule byhomologous, or site-specific, recombination. In another embodiment, TR21polynucleotides and corresponding polypeptides may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of TR21 may be recombined with one or more components,motifs, sections, parts, domains, fragments, etc. of one or moreheterologous molecules. In preferred embodiments, the heterologousmolecules are include, but are not limited to, TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), TRAIL, AIM-II (International Publication No. WO 97/34911),APRIL (J. Exp. Med. 188(6):1185-1190 (1998)), endokine-alpha(International Publication No. WO 98/07880), Neutrokine alpha(International Publication No. WO98/18921), OPG, OX40, and nerve growthfactor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2(International Publication No. WO 96/34095), DR3 (InternationalPublication No. WO 97/33904), DR4 (International Publication No. WO98/32856), TR5 (International Publication No. WO 98/30693), TR6(International Publication No. WO 98/30694), TR7 (InternationalPublication No. WO 98/41629), TRANK, TR9 (International Publication No.WO 98/56892), 312C2 (International Publication No. WO 98/06842), andTR12, and soluble forms CD154, CD70, and CD153. In further preferredembodiments, the heterologous molecules are any member of the TNFfamily.

Proteins and other compounds which bind the TR21 domains are alsocandidate agonists and antagonists according to the present invention.Such binding compounds can be “captured” using the yeast two-hybridsystem (Fields and Song, Nature 340:245-246 (1989)). A modified versionof the yeast two- hybrid system has been described by Roger Brent andhis colleagues (Gyuris, Cell 75:791-803 (1993); Zervos et al., Cell72:223-232 (1993)). Preferably, the yeast two-hybrid system is usedaccording to the present invention to capture compounds which bind tothe extracellular domain, intracellular, transmembrane, and the cysteinerich domain of TR21. Such compounds are good candidate agonists andantagonists of the present invention.

For example, using the two-hybrid assay described above, theextracellular or intracellular domain of the TR21, or a portion thereof(e.g., the cysteine rich domain), may be used to identify cellularproteins which interact with TR21 the receptor in vivo. Such an assaymay also be used to identify ligands with potential agonistic orantagonistic activity of TR21 receptor function. This screening assayhas previously been used to identify protein which interact with thecytoplasmic domain of the murine TNF-RII and led to the identificationof two receptor associated proteins. Rothe et al., Cell 78:681 (1994).Such proteins and amino acid sequences which bind to the cytoplasmicdomain of the TR21 are good candidate agonist and antagonist of thepresent invention.

Other screening techniques include the use of cells which express thepolypeptide of the present invention (for example, transfected CHOcells) in a system which measures extracellular pH changes caused byreceptor activation, for example, as described in Science, 246:181-296(1989). In another example, potential agonists or antagonists may becontacted with a cell which expresses the polypeptide of the presentinvention and a second messenger response, e.g., signal transduction maybe measured to determine whether the potential antagonist or agonist iseffective.

Agonists according to the present invention include naturally occurringand synthetic compounds such as, for example, TNF family ligand peptidefragments, transforming growth factor, neurotransmitters (such asglutamate, dopamine, N-methyl-D-aspartate), tumor suppressors (p53),cytolytic T cells and antimetabolites. Preferred agonists includechemotherapeutic drugs such as, for example, cisplatin, doxorubicin,bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate andvincristine. Others include ethanol and -amyloid peptide. (Science267:1457-1458 (1995)).

Preferred agonists are fragments of TR21 polypeptides of the inventionwhich stimulate lymphocyte (e.g., B cell) proliferation, differentiationand/or activation. Further preferred agonists include polyclonal andmonoclonal antibodies raised against the TR21 polypeptides of theinvention, or a fragment thereof. Such agonist antibodies raised againsta TNF-family receptor are disclosed in Tartaglia et al., Proc. Natl.Acad. Sci. USA 88:9292-9296 (1991); and Tartaglia et al., J. Biol. Chem.267:4304-4307(1992). See, also, PCT Application WO 94/09137.

In an additional embodiment, immunoregulatory molecules such as, forexample, IL2, IL3, IL4, ILS, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD4OL, IFN-gamma and TNF-alpha, may be used as agonists ofTR21 polypeptides of the invention which stimulate lymphocyte (e.g., Bcell) proliferation, differentiation and/or activation. In a specificembodiment, IL4 and/or IL10 are used to enhance the TR21-mediatedproliferation of B cells.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959 each of which is incorporated by reference herein inits entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well-known techniquesto prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form that while allowing for an exchange of components withthe immediate extracellular environment does not allow the introducedcells to be recognized by the host immune system.

In yet another embodiment of the invention, the activity of TR21polypeptide can be reduced using a “dominant negative.” To this end,constructs which encode, for example, defective TR21 polypeptide, suchas, for example, mutants lacking all or a portion of the TNF-conserveddomain, can be used in gene therapy approaches to diminish the activityof TR21 on appropriate target cells. For example, nucleotide sequencesthat direct host cell expression of TR21 polypeptide in which all or aportion of the TNFR-conserved domain is altered or missing can beintroduced into monocytic cells or other cells or tissues (either by invivo or ex vivo gene therapy methods described herein or otherwise knownin the art). Alternatively, targeted homologous recombination can beutilized to introduce such deletions or mutations into the subject'sendogenous TR21 gene in monocytes. The engineered cells will expressnon-functional TR21 polypeptides (i.e., a receptor (e.g., multimer) thatmay be capable of binding, but which is incapable of inducing signalstransduction).

Diagnostic Assays

The compounds of the present invention are useful for diagnosis ortreatment of various immune system-related disorders in mammals,preferably humans. Such disorders include but are not limited to tumors(e.g., B cell and monocytic cell leukemias and lymphomas) and tumormetastasis, infections by bacteria, viruses and other parasites,immunodeficiencies, inflammatory diseases, lymphadenopathy, autoimmunediseases, and graft versus host disease.

TR21 is expressed in B cells and spleen. For a number of immunesystem-related disorders, substantially altered (increased or decreased)levels of TR21 gene expression can be detected in immune system tissueor other cells or bodily fluids (e.g., sera, plasma, urine, synovialfluid or spinal fluid) taken from an individual having such a disorder,relative to a “standard” TR21 gene expression level, that is, the TR21expression level in immune system tissues or bodily fluids from anindividual not having the immune system disorder. Thus, the inventionprovides a diagnostic method useful during diagnosis of an immune systemdisorder, which involves measuring the expression level of the geneencoding the TR21 polypeptide in immune system tissue or other cells orbody fluid from an individual and comparing the measured gene expressionlevel with a standard TR21 gene expression level, whereby an increase ordecrease in the gene expression level(s) compared to the standard isindicative of an immune system disorder or normal activation,proliferation, differentiation, and/or death.

In particular, it is believed that certain tissues in mammals withcancer of cells or tissue of the immune system express significantlyenhanced or reduced levels of normal or altered TR21 polypeptide andmRNA encoding the TR21 polypeptide when compared to a corresponding“standard” level. Further, it is believed that enhanced or depressedlevels of the TR21 polypeptide can be detected in certain body fluids(e.g., sera, plasma, urine, and spinal fluid) or cells or tissue frommammals with such a cancer when compared to sera from mammals of thesame species not having the cancer.

For example, as disclosed herein, TR21 are expressed in B cells.Accordingly, polynucleotides of the invention (e.g., polynucleotidesequences complementary to all or a portion of TR21 mRNA) and antibodies(and antibody fragments) directed against the polypeptides of theinvention may be used to quantitate or qualitate concentrations of cellsof B cell lineage (e.g., B cell leukemia cells) expressing TR21 on theircell surfaces. These antibodies additionally have diagnosticapplications in detecting abnormalities in the level of TR21 geneexpression, or abnormalities in the structure and/or temporal, tissue,cellular, or subcellular location of TR21. These diagnostic assays maybe performed in vivo or in vitro, such as, for example, on bloodsamples, biopsy tissue or autopsy tissue.

Thus, the invention provides a diagnostic method useful during diagnosisof a immune system disorder, including cancers of this system, whichinvolves measuring the expression level of the gene encoding the TR21polypeptide in immune system tissue or other cells or body fluid from anindividual and comparing the measured gene expression level with astandard TR21 gene expression level, whereby an increase or decrease inthe gene expression level compared to the standard is indicative of animmune system disorder.

Where a diagnosis of a disorder in the immune system, includingdiagnosis of a tumor, has already been made according to conventionalmethods, the present invention is useful as a prognostic indicator,whereby patients exhibiting enhanced or depressed TR21 gene expressionwill experience a worse clinical outcome relative to patients expressingthe gene at a level nearer the standard level.

By “assaying the expression level of the gene encoding the TR21polypeptide” is intended qualitatively or quantitatively measuring orestimating the level of the TR21 polypeptide or the level of the mRNAencoding the TR21 polypeptide in a first biological sample eitherdirectly (e.g., by determining or estimating absolute protein level ormRNA level) or relatively (e.g., by comparing to the TR21 polypeptidelevel or mRNA level in a second biological sample). Preferably, the TR21polypeptide level or mRNA level in the first biological sample ismeasured or estimated and compared to a standard TR21 polypeptide levelor mRNA level, the standard being taken from a second biological sampleobtained from an individual not having the disorder or being determinedby averaging levels from a population of individuals not having adisorder of the immune system. As will be appreciated in the art, once astandard TR21 polypeptide level or mRNA level is known, it can be usedrepeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, cell line, tissue culture, or other source containingTR21 receptor protein (including portions thereof) or mRNA. Asindicated, biological samples include body fluids (such as sera, plasma,urine, synovial fluid and spinal fluid) which contain free extracellulardomains of the TR21 polypeptide, immune system tissue, and other tissuesources found to express complete or free extracellular domain of theTR21 receptor. Methods for obtaining tissue biopsies and body fluidsfrom mammals are well known in the art. Where the biological sample isto include mRNA, a tissue biopsy is the preferred source.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the TR21 polypeptide are then assayed using anyappropriate method. These include Northern blot analysis, Si nucleasemapping, the polymerase chain reaction (PCR), reverse transcription incombination with the polymerase chain reaction (RT-PCR), and reversetranscription in combination with the ligase chain reaction (RT-LCR).

The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of TR21 receptorprotein, or the soluble form thereof, in a biological sample (e.g.,cells and tissues), including determination of normal and abnormallevels of polypeptides. Thus, for instance, a diagnostic assay inaccordance with the invention for detecting over-expression of TR21, orsoluble form thereof, compared to normal control tissue samples may beused to detect the presence of tumors, for example. Assay techniquesthat can be used to determine levels of a protein, such as a TR21protein of the present invention, or a soluble form thereof, in a samplederived from a host are well-known to those of skill in the art. Suchassay methods include radioimmunoassays, competitive-binding assays,Western Blot analysis and ELISA assays. Assaying TR21 protein levels ina biological sample can occur using any art-known method.

Assaying TR21 polypeptide levels in a biological sample can occur usingantibody-based techniques. For example, TR21 polypeptide expression intissues can be studied with classical immunohistological methods(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell . Biol. 105:3087-3096 (1987)). Other antibody-basedmethods useful for detecting TR21 polypeptide gene expression includeimmunoassays, such as the enzyme linked immunosorbent assay (ELISA) andthe radioimmunoassay (RIA). Suitable antibody assay labels are known inthe art and include enzyme labels, such as, glucose oxidase, andradioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹²In), and technetium (^(99m)Tc), and fluorescentlabels, such as fluorescein and rhodamine, and biotin.

The tissue or cell type to be analyzed will generally include thosewhich are known, or suspected, to express the TR21 gene (such as, forexample, cells of B cell lineage and the spleen) or cells or tissuewhich are known, or suspected, to express the TR21 ligand gene (such as,for example, cells of monocytic lineage). The protein isolation methodsemployed herein may, for example, be such as those described in Harlowand Lane (Harlow, E. and Lane, D., 1988, “Antibodies: A LaboratoryManual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.),which is incorporated herein by reference in its entirety. The isolatedcells can be derived from cell culture or from a patient. The analysisof cells taken from culture may be a necessary step in the assessment ofcells that could be used as part of a cell-based gene therapy techniqueor, alternatively, to test the effect of compounds on the expression ofthe TR21 gene or TR21 ligand gene.

For example, antibodies, or fragments of antibodies, such as thosedescribed herein, may be used to quantitatively or qualitatively detectthe presence of TR21 gene products or conserved variants or peptidefragments thereof. This can be accomplished, for example, byimmunofluorescence techniques employing a fluorescently labeled antibodycoupled with light microscopic, flow cytometric, or fluorimetricdetection.

The antibodies (or fragments thereof), TR21 polypeptides, and/or TR21ligands (e.g., Neutrokine-alpha) of the present invention may,additionally, be employed histologically, as in immunofluorescence,immunoelectron microscopy or non-immunological assays, for in situdetection of TR21 gene products or conserved variants or peptidefragments thereof, or for TR21 binding to TR21 ligand. In situ detectionmay be accomplished by removing a histological specimen from a patient,and applying thereto a labeled antibody or TR21 polypeptide of thepresent invention. The antibody (or fragment) or TR21 polypeptide ispreferably applied by overlaying the labeled antibody (or fragment) ontoa biological sample. Through the use of such a procedure, it is possibleto determine not only the presence of the TR21 gene product, orconserved variants or peptide fragments, or TR21 polypeptide binding,but also its distribution in the examined tissue. Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

Immunoassays and non-immunoassays for TR21 gene products or conservedvariants or peptide fragments thereof will typically comprise incubatinga sample, such as a biological fluid, a tissue extract, freshlyharvested cells, or lysates of cells which have been incubated in cellculture, in the presence of a detectably labeled antibody capable ofbinding TR21 gene products or conserved variants or peptide fragmentsthereof, and detecting the bound antibody by any of a number oftechniques well-known in the art.

Immunoassays and non-immunoassays for TR21 ligand gene products orconserved variants or peptide fragments thereof will typically compriseincubating a sample, such as a biological fluid, a tissue extract,freshly harvested cells, or lysates of cells which have been incubatedin cell culture, in the presence of a detectable or labeled TR21polypeptide capable of identifying TR21 ligand gene products orconserved variants or peptide fragments thereof, and detecting the boundTR21 polypeptide by any of a number of techniques well-known in the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled anti-TR21 antibody ordetectable TR21 polypeptide. The solid phase support may then be washedwith the buffer a second time to remove unbound antibody or polypeptide.Optionally the antibody is subsequently labeled. The amount of boundlabel on solid support may then be detected by conventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-TR21 antibody or TR21polypeptide may be determined according to well-known methods. Thoseskilled in the art will be able to determine operative and optimal assayconditions for each determination by employing routine experimentation.

In addition to assaying TR21 polypeptide levels or polynucleotide levelsin a biological sample obtained from an individual, TR21 polypeptide orpolynucleotide can also be detected in vivo by imaging. For example, inone embodiment of the invention, TR21 polypeptide is used to imagemonocytic leukemias or lymphomas. In another embodiment, TR21polynucleotides of the invention and/or anti-TR21 antibodies (e.g.,polynucleotides complementary to all or a portion of TR21 mRNA) are usedto image B cell leukemias or lymphomas.

Antibody labels or markers for in vivo imaging of TR21 polypeptideinclude those detectable by X-radiography, NMR, MRI, CAT-scans or ESR.For X-radiography, suitable labels include radioisotopes such as bariumor cesium, which emit detectable radiation but are not overtly harmfulto the subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma. Where in vivo imaging is used to detect enhanced levels ofTR21 polypeptide for diagnosis in humans, it may be preferable to usehuman antibodies or “humanized” chimeric monoclonal antibodies. Suchantibodies can be produced using techniques described herein orotherwise known in the art. For example methods for producing chimericantibodies are known in the art. See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature314:268 (1985).

Additionally, any TR21 polypeptide whose presence can be detected, canbe administered. For example, TR21 polypeptides labeled with aradio-opaque or other appropriate compound can be administered andvisualized in vivo, as discussed, above for labeled antibodies. Furthersuch TR21 polypeptides can be utilized for in vitro diagnosticprocedures.

A TR21 polypeptide-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for immune systemdisorder. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ^(99m)Tc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain TR21 protein. In vivotumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

With respect to antibodies, one of the ways in which the anti-TR21antibody can be detectably labeled is by linking the same to an enzymeand using the linked product in an enzyme immunoassay (EIA) (Voller, A.,“The Enzyme Linked Immunosorbent Assay (ELISA)”, 1978, DiagnosticHorizons 2:1-7, Microbiological Associates Quarterly Publication,Walkersville, Md.); Voller et al., J. Clin. Pathol. 31:507-520 (1978);Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980,Enzyme Immunoassay, CRC Press, Boca Raton, Fla.; Ishikawa, E. et al.,(eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme whichis bound to the antibody will react with an appropriate substrate,preferably a chromogenic substrate, in such a manner as to produce achemical moiety which can be detected, for example, byspectrophotometric, fluorimetric or by visual means. Enzymes which canbe used to detectably label the antibody include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate,dehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. Additionally, the detection canbe accomplished by colorimetric methods which employ a chromogenicsubstrate for the enzyme. Detection may also be accomplished by visualcomparison of the extent of enzymatic reaction of a substrate incomparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect TR21 through the use of aradioimmunoassay (RIA) (see, for example, Weintraub, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March, 1986, which is incorporated byreference herein). The radioactive isotope can be detected by meansincluding, but not limited to, a gamma counter, a scintillation counter,or autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wavelength, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Chromosome Assays

The nucleic acid molecules of the present invention are also valuablefor chromosome identification.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a TR21 receptor gene. This canbe accomplished using a variety of well-known techniques and libraries,which generally are available commercially. The genomic DNA is then usedfor in situ chromosome mapping using well-known techniques for thispurpose.

In addition, in some cases, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3′ untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes.

Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bp. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man, available on line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

ILLUSTRATIVE EXAMPLES Example 1 Protein Fusions of TR21

TR21 polypeptides of the invention are optionally fused to otherproteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of TR21 polypeptides to His-tag,HA-tag, protein A, IgG domains, and maltose binding protein facilitatespurification. (See EP A 394,827; Traunecker, et al., Nature 331:84-86(1988)). Similarly, fusion to IgG-1, IgG-3, and albumin increases thehalf-life time in vivo. Nuclear localization signals fused to TR21polypeptides can target the protein to a specific subcellularlocalization, while covalent heterodimer or homodimers can increase ordecrease the activity of a fusion protein. Fusion proteins can alsocreate chimeric molecules having more than one function. Finally, fusionproteins can increase solubility and/or stability of the fused proteincompared to the non-fused protein. All of the types of fusion proteinsdescribed above can be made using techniques known in the art or byusing or routinely modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow (SEQ ID NO:3). These primers also preferably contain convenientrestriction enzyme sites that will facilitate cloning into an expressionvector, preferably a mammalian expression vector.

For example, if the pC4 (Accession No. 209646) expression vector isused, the human Fc portion can be ligated into the BamHI cloning site.Note that the 3′ BamHI site should be destroyed. Next, the vectorcontaining the human Fc portion is re-restricted with BamHI, linearizingthe vector, and TR21 polynucleotide is ligated into this BamHI site.Note that the polynucleotide is cloned without a stop codon otherwise afusion protein will not be produced.

If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See e.g., WO 96/34891.)

Human IgG Fc Region:

(SEQ ID NO: 3)GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 2 Isolation of Antibody Fragments Directed Against Polypeptidesof the Present Invention from a Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a large library of antibody fragments which contain reactivitiesagainst polypeptides of the present invention to which the donor may ormay not have been exposed (see e.g., U.S. Pat. No. 5,885,793incorporated herein in its entirety by reference).

Rescue of the Library

A library of scFvs is constructed from the RNA of human PBLs asdescribed in WO92/01047. To rescue phages displaying antibody fragments,approximately 10⁹ E. coli harboring the phagemid are used to inoculate50 ml of 2xTY containing 1% glucose and 100 ug/ml of ampicillin(2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of thisculture is used to innoculate 50 ml of 2xTY-AMP-GLU, 2×108 TU of Δ gene3 helper phage (M13 Δ gene III, see WO92/01047) are added and theculture incubated at 37° C. for 45 minutes without shaking and then at37° C. for 45 minutes with shaking. The culture is centrifuged at 4000r.p.m. for 10 minutes and the pellet resuspended in 2 liters of 2xTYcontaining 100 ug/ml ampicillin and 50 ug/ml kanamycin and grownovernight. Phages are prepared as described in WO92/01047.

M13 Δ gene III is prepared as follows: M13 A gene III helper phage doesnot encode gene III protein, hence the phage(mid) displaying antibodyfragments have a greater avidity of binding to antigen. Infectious M13 Δgene III particles are made by growing the helper phage in cellsharboring a pUC19 derivative supplying the wild type gene III proteinduring phage morphogenesis. The culture is incubated for 1 hour at 37°C. without shaking and then for a further hour at 37° C. with shakingCells are pelleted (IEC-Centra 8, 4000 revs/min for 10 min), resuspendedin 300 ml 2xTY broth containing 100 ug ampicillin/ml and 25 ugkanamycin/ml (2xTY-AMP-KAN) and grown overnight, shaking at 37° C. Phageparticles are purified and concentrated from the culture medium by twoPEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS andpassed through a 0.45 um filter (Minisart NML; Sartorius) to give afinal concentration of approximately 10¹³ transducing units/ml(ampicillin-resistant clones).

Panning of the Library

Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100mg/ml or 10 mg/ml of a polypeptide of the present invention. Tubes areblocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 timesin PBS. Approximately 10¹³ TU of phage are applied to the tube andincubated for 30 minutes at room temperature tumbling on an over andunder turntable and then left to stand for another 1.5 hours. Tubes arewashed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage areeluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes onan under and over turntable after which the solution is immediatelyneutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phages are then usedto infect 10 ml of mid-log E. coli TG1 by incubating eluted phages withbacteria for 30 minutes at 37° C. The E. coli are then plated on TYEplates containing 1% glucose and 100 ug/ml ampicillin. The resultingbacterial library is then rescued with Δ gene III helper phage asdescribed above to prepare phage for a subsequent round of selection.This process is then repeated for a total of 4 rounds of affinitypurification with tube-washing increased to 20 times with PBS, 0.1%Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders

Eluted phage from the 3rd and 4th rounds of selection are used to infectE. coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) fromsingle colonies for assay. ELISAs are performed with microtiter platescoated with either 10 pg/ml of the polypeptide of the present inventionin 50 mM bicarbonate pH 9.6. Clones positive in ELISA are furthercharacterized by PCR fingerprinting (see e.g., WO92/01047) and then bysequencing.

Example 3 Production of a TR21 Antibody a) Hybridoma Technology

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing TR21 are administered to an animal to inducethe production of sera containing polyclonal antibodies. In a preferredmethod, a preparation of TR21 protein is prepared and purified to renderit substantially free of natural contaminants. Such a preparation isthen introduced into an animal in order to produce polyclonal antiseraof greater specific activity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or protein binding fragments thereof). Suchmonoclonal antibodies can be prepared using hybridoma technology.(Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol.6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerlinget al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,pp. 563-681 (1981).) In general, such procedures involve immunizing ananimal (preferably a mouse) with TR21 polypeptide or, more preferably,with a secreted TR21 polypeptide-expressing cell. Such cells may becultured in any suitable tissue culture medium; however, it ispreferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 g/l of nonessential amin acids, about1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP2O), available from the ATCC™.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the TR21 polypeptide.

Alternatively, additional antibodies capable of binding to TR21polypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodywhich binds to a second antibody. In accordance with this method,protein specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the TR21protein-specific antibody can be blocked by TR21. Such antibodiescomprise anti-idiotypic antibodies to the TR21 protein-specific antibodyand can be used to immunize an animal to induce formation of furtherTR21 protein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). Alternatively, secreted TR21protein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. (See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985)).

b) Isolation of Antibody Fragments Directed Against TR21 from a Libraryof scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a large library of antibody fragments which contain reactivitiesagainst TR21 to which the donor may or may not have been exposed (seee.g., U.S. Pat. No. 5,885,793 incorporated herein in its entirety byreference).

Rescue of the Library

A library of scFvs is constructed from the RNA of human PBLs asdescribed in W092/01047. To rescue phages displaying antibody fragments,approximately 10⁹ E. coli harboring the phagemid are used to inoculate50 ml of 2xTY containing 1% glucose and 100 ug/ml of ampicillin(2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking Five ml of thisculture is used to innoculate 50 ml of 2xTY-AMP-GLU, 2×10⁸ TU of deltagene 3 helper (M13 delta gene III, see WO92/01047) are added and theculture incubated at 37° C. for 45 minutes without shaking and then at37° C. for 45 minutes with shaking. The culture is centrifuged at 4000r.p.m. for 10 min. and the pellet resuspended in 2 liters of 2xTYcontaining 100 ug/ml ampicillin and 50 ug/ml kanamycin and grownovernight. Phages are prepared as described in WO92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (IEC-Centra 8, 4000 revs/min for 10min), resuspended in 300 ml 2xTY broth containing 100 ug ampicillin/mland 25 ug kanamycin/ml (2xTY-AMP-KAN) and grown overnight, shaking at37° C. Phage particles are purified and concentrated from the culturemedium by two PEG-precipitations (Sambrook et al., 1990), resuspended in2 ml PBS and passed through a 0.45 um filter (Minisart NML; Sartorius)to give a final concentration of approximately 10¹³ transducing units/ml(ampicillin-resistant clones).

Panning of the Library

Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100ug/ml or 10 ug/ml of a polypeptide of the present invention. Tubes areblocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 timesin PBS. Approximately 10¹³ TU of phage is applied to the tube andincubated for 30 minutes at room temperature tumbling on an over andunder turntable and then left to stand for another 1.5 hours. Tubes arewashed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage areeluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes onan under and over turntable after which the solution is immediatelyneutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phages are then usedto infect 10 ml of mid-log E. coli TG1 by incubating eluted phage withbacteria for 30 minutes at 37° C. The E. coli are then plated on TYEplates containing 1% glucose and 100 μg/ml ampicillin. The resultingbacterial library is then rescued with delta gene 3 helper phage asdescribed above to prepare phage for a subsequent round of selection.This process is then repeated for a total of 4 rounds of affinitypurification with tube-washing increased to 20 times with PBS, 0.1%Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders

Eluted phages from the 3rd and 4th rounds of selection are used toinfect E. coli HB 2151 and soluble scFv is produced (Marks, et al.,1991) from single colonies for assay. ELISAs are performed withmicrotiter plates coated with either 10 pg/ml of the polypeptide of thepresent invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISAare further characterized by PCR fingerprinting (see e.g., WO92/01047)and then by sequencing.

Example 4 Method of Detecting Abnormal Levels of TR21 in a BiologicalSample

TR21 polypeptides can be detected in a biological sample, and if anincreased or decreased level of TR21 is detected, this polypeptide is amarker for a particular phenotype. Methods of detection are numerous,and thus, it is understood that one skilled in the art can modify thefollowing assay to fit their particular needs.

For example, antibody-sandwich ELISA assays are used to detect TR21 in asample, preferably a biological sample. Wells of a microtiter plate arecoated with specific antibodies to TR21, at a final concentration of 0.2to 10 ug/ml. The antibodies are either monoclonal or polyclonal and areproduced using technique known in the art. The wells are blocked so thatnon-specific binding of TR21 to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining TR21. Preferably, serial dilutions of the sample should beused to validate results. The plates are then washed three times withdeionized or distilled water to remove unbound TR21.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25-400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate(NPP) substrate solution is then added to each well and incubated 1 hourat room temperature to allow cleavage of the substrate and fluorescence.The fluorescence is measured using a microtiter plate reader. A standardcurve is prepared using the experimental results from serial dilutionsof a control sample with the sample concentration plotted on the X-axis(log scale) and fluorescence or absorbance on the Y-axis (linear scale).The TR21 polypeptide concentration in a sample is then interpolatedusing the standard curve based on the measured fluorescence of thatsample.

Example 5 Method of Treating Decreased Levels of TR21

The present invention relates to a method for treating an individual inneed of a decreased level of TR21 biological activity in the bodycomprising, administering to such an individual a composition comprisinga therapeutically effective amount of TR21 antagonist. Preferredantagonists for use in the present invention are TR21-specificantibodies.

Moreover, it will be appreciated that conditions caused by a decrease inthe standard or normal expression level of TR21 in an individual can betreated by administering TR21, preferably in a soluble and/or secretedform. Thus, the invention also provides a method of treatment of anindividual in need of an increased level of TR21 polypeptide comprisingadministering to such an individual a pharmaceutical compositioncomprising an amount of TR21 to increase the biological activity levelof TR21 in such an individual.

For example, a patient with decreased levels of TR21 polypeptidereceives a daily dose 0.1-100 ug/kg of the polypeptide for sixconsecutive days. Preferably, the polypeptide is in a soluble and/orsecreted form.

Example 6 Method of Treating Increased Levels of TR21

The present invention also relates to a method for treating anindividual in need of an increased level of TR21 biological activity inthe body comprising administering to such an individual a compositioncomprising a therapeutically effective amount of TR21 or an agonistthereof.

Antisense technology is used to inhibit production of TR21. Thistechnology is one example of a method of decreasing levels of TR21polypeptide, preferably a soluble and/or secreted form, due to a varietyof etiologies, such as cancer.

For example, a patient diagnosed with abnormally increased levels ofTR21 is administered intravenously antisense polynucleotides at 0.5,1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeatedafter a 7-day rest period if the is determined to be well tolerated.

Example 7

Method of Treatment Using Gene Therapy—Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing soluble and/or mature TR21 polypeptides, onto a patient.Generally, fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin) is added. The flasks are then incubated at 37° C. forapproximately one week.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding TR21 can be amplified using PCR primers whichcorrespond to the 5′ and 3′ end encoding sequences respectively.Preferably, the 5′ primer contains an EcoRI site and the 3′ primerincludes a HindIII site. Equal quantities of the Moloney murine sarcomavirus linear backbone and the amplified EcoRI and HindIII fragment areadded together, in the presence of T4 DNA ligase. The resulting mixtureis maintained under conditions appropriate for ligation of the twofragments. The ligation mixture is then used to transform E. coli HB101,which are then plated onto agar containing kanamycin for the purpose ofconfirming that the vector contains properly inserted TR21.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the TR21 gene is then added to the media and the packagingcells transduced with the vector. The packaging cells now produceinfectious viral particles containing the TR21 gene (the packaging cellsare now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether TR21 proteinis produced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 8 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) TR21 sequences into an animal to increase ordecrease the expression of the TR21 polypeptide. The TR21 polynucleotidemay be operatively linked to a promoter or any other genetic elementsnecessary for the expression of the TR21 polypeptide by the targettissue. Such gene therapy and delivery techniques and methods are knownin the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos.5,693,622, 5,705,151, 5,580,859; Tabata H. et al., Cardiovasc. Res.35:470-479 (1997); Chao J. et al., Pharmacol. Res. 35:517-522 (1997);Wolff J. A. Neuromuscul. Disord. 7:314-318 (1997); Schwartz B. et al.,Gene Ther. 3:405-411 (1996); Tsurumi Y. et al., Circulation 94:3281-3290(1996) (incorporated herein by reference).

The TR21 polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The TR21 polynucleotide constructscan be delivered in a pharmaceutically acceptable liquid or aqueouscarrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, LIPOFECTIN™ or precipitating agentsand the like. However, the TR21 polynucleotides may also be delivered inliposome formulations (such as those taught in Feigner P. L., et al.Ann. NY Acad. Sci. 772:126-139 (1995), and Abdallah B., et al. Biol.Cell 85(1):1-7 (1995)) which can be prepared by methods well known tothose skilled in the art.

The TR21 polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication. Anystrong promoter known to those skilled in the art can be used fordriving the expression of DNA. Unlike other gene therapy techniques, onemajor advantage of introducing naked nucleic acid sequences into targetcells is the transitory nature of the polynucleotide synthesis in thecells. Studies have shown that non-replicating DNA sequences can beintroduced into cells to provide production of the desired polypeptidefor periods of up to six months.

The TR21 polynucleotide construct can be delivered to the interstitialspace of tissues within the animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked TR21 polynucleotide injection, an effective dosage amountof DNA or RNA will be in the range of from about 0.05 g/kg body weightto about 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked TR21polynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected TR21 polynucleotide in muscle invivo is determined as follows. Suitable TR21 template DNA for productionof mRNA coding for TR21 polypeptide is prepared in accordance with astandard recombinant DNA methodology. The template DNA, which may beeither circular or linear, is either used as naked DNA or complexed withliposomes. The quadriceps muscles of mice are then injected with variousamounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The TR21 template DNA is injected in 0.1 ml of carrier in a1 cc syringe through a 27 gauge needle over one minute, approximately0.5 cm from the distal insertion site of the muscle into the knee andabout 0.2 cm deep. A suture is placed over the injection site for futurelocalization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 umcross-section of the individual quadriceps muscles is histochemicallystained for TR21 protein expression. A time course for TR21 proteinexpression may be done in a similar fashion except that quadriceps fromdifferent mice are harvested at different times. Persistence of TR21 DNAin muscle following injection may be determined by Southern blotanalysis after preparing total cellular DNA and HIRT supernatants frominjected and control mice. The results of the above experimentation inmice can be use to extrapolate proper dosages and other treatmentparameters in humans and other animals using TR21 naked DNA.

Example 9 Gene Therapy Using Endogenous TR21 Gene

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous TR21 sequence with apromoter via homologous recombination as described, for example, in U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International PublicationNumber WO 96/29411; International Publication Number WO 94/12650; Kolleret al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra etal., Nature 342:435-438 (1989). This method involves the activation of agene which is present in the target cells, but which is not expressed inthe cells, or is expressed at a lower level than desired. Polynucleotideconstructs are made which contain a promoter and targeting sequences,which are homologous to the 5′ non-coding sequence of endogenous TR21,flanking the promoter. The targeting sequence will be sufficiently nearthe 5′ end of TR21 so the promoter will be operably linked to theendogenous sequence upon homologous recombination. The promoter and thetargeting sequences can be amplified using PCR. Preferably, theamplified promoter contains distinct restriction enzyme sites on the 5′and 3′ ends. Preferably, the 3′ end of the first targeting sequencecontains the same restriction enzyme site as the 5′ end of the amplifiedpromoter and the 5′ end of the second targeting sequence contains thesame restriction site as the 3′ end of the amplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenousTR21 sequence. This results in the expression of TR21 in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the TR21 locus, plasmid pUC18(MBI Fermentas, Amherst, NY) is digested with HindIII. The CMV promoteris amplified by PCR with an XbaI site on the 5′ end and a BamHI site onthe 3′end. Two TR21 non-coding sequences are amplified via PCR: one TR21non-coding sequence (TR21 fragment 1) is amplified with a HindIII siteat the 5′ end and an Xba site at the 3′end; the other TR21 non-codingsequence (TR21 fragment 2) is amplified with a BamHI site at the 5′endand a HindIII site at the 3′end. The CMV promoter and TR21 fragments aredigested with the appropriate enzymes (CMV promoter—XbaI and BamHI; TR21fragment 1—XbaI; TR21 fragment 2—BamHI) and ligated together. Theresulting ligation product is digested with HindIII, and ligated withthe HindIII-digested pUC18 plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5×10⁶ cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and250-300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37° C. The following day, the media is aspiratedand replaced with 10 ml of fresh media and incubated for a further 16-24hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 10 Bioassay for the Effect of TR21 Polypeptides, Agonists, orAntagonists on Hematopoietic Progenitor Cells and/or Differentiation

Mouse bone marrow cells may be used as target cells to examine theeffect of TR21 polypeptides of the invention on hematopoietic progenitorcells and/or differentiation. Briefly, unfractionated bone marrow cellsare first washed 2× with a serum-free IMDM that is supplemented with 10%(V/V) BIT (Bovine serum albumin, Insulin and Transferrin supplement fromStem Cell Technologies, Vancouver, Canada). The washed cells are thenresuspended in the same growth medium and plated in the 96-well tissueculture plate (5×10⁴ cells/well) in 0.2 ml of the above medium in thepresence or absence of cytokines and TR21. Stem cell factor (SCF) andIL-3 are included as positive mediators of cell proliferation. Cells areallowed to grow in a low oxygen environment (5% CO₂, 7% O², and 88% N₂)tissue culture incubator for 6 days. On the sixth day, 0.5 μCi ofTritiated thymidine is added to each well and incubation is continuedfor an additional 16-18 hours, at which point the cells are harvested.The level of radioactivity incorporated into cellular DNA is determinedby scintillation spectrometry and reflects the amount of cellproliferation.

The studies described in this example test the activity of TR21polypeptides of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of TR21polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21. Potential agonists would be expected to inhibit hematopoietic cellproliferation in the presence of SCF and/or IL3 and/or to increase theinhibition of cell proliferation in the presence of cytokines and TR21in this assay. Potential antagonists would be expected to reduce theinhibition of cell proliferation in the presence of cytokines and TR21in this assay.

Example 11 Bioassay for the Effect of TR21 Polypeptides, Agonists orAntagonists on IL-3 and SCF Stimulated Proliferation and Differentiationof Hematopoietic Progenitor Cells

To determine if TR21 polypeptides of the invention inhibit specifichematopoietic lineages, mouse bone marrow cells are first washed 2× witha serum-free IMDM that is supplemented with 10% (V/V) BIT (Bovine serumalbumin, Insulin and Transferrin supplement from Stem Cell Technologies,Vancouver, Canada). The washed cells are then resuspended in the samegrowth medium and plated in the 96-well tissue culture plate (5×10⁴cells/well) in 0.2 ml of the above medium in the presence of IL-3 (1ng/ml) plus SCF (5 ng/ml) with or without TR21. Cells are allowed togrow in a low oxygen environment (5% CO₂, 7% O², and 88% N₂) tissueculture incubator, and after 7 days, analyzed for expression ofdifferentiation antigens by staining with various monoclonal antibodiesand FACScan.

The studies described in this example test the activity of TR21polypeptides of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity of TR21polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofTR21. Potential agonists tested in this assay would be expected toinhibit cell proliferation in the presence of cytokines and/or toincrease the inhibition of cell proliferation in the presence ofcytokines and TR21. Potential antagonists tested in this assay would beexpected to reduce the inhibition of cell proliferation in the presenceof cytokines and TR21.

Example 12 Effect of TR21 on IL-3 and SCF Stimulated Proliferation andDifferentiation of Lin-Population of Bone Marrow Cells

A population of mouse bone marrow cells enriched in primitivehematopoietic progenitors can be obtained using a negative selectionprocedure, where the committed cells of most of the lineages are removedusing a panel of monoclonal antibodies (anti cd11b, CD4, CD8, CD45R andGr-1 antigens) and magnetic beads. The resulting population of cells(lineage depleted cells) are plated (5×10⁴ cells/ml) in the presence orabsence of TR21 polypeptide of the invention (in a range ofconcentrations) in a growth medium supplemented with IL-3 (5 ng/ml) plusSCF (100 ng/ml). After seven days of incubation at 37° C. in ahumidified incubator (5% CO₂, 7% O₂, and 88% N₂ environment), cells areharvested and assayed for the HPP-CFC, and immature progenitors. Inaddition, cells are analyzed for the expression of certaindifferentiation antigens by FACScan. Colony data is expressed as meannumber of colonies+/−SD) and are obtained from assays performed in sixdishes for each population of cells.

Example 13

Assays to Detect Stimulation or Inhibition of B Cell Proliferation andDifferentiation

Generation of functional humoral immune responses requires both solubleand cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL5, IL6,IL-7, IL10, IL-13, IL14 and IL15. Interestingly, these signals are bythemselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.One of the most well studied classes of B-cell co-stimulatory proteinsis the TNF-superfamily. Within this family CD40, CD27, and CD30 alongwith their respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

In Vitro Assay

Purified TR21 polypeptides of the invention (e.g., soluble TR21) oragonists or antagonists thereof, are assessed for their ability toinduce activation, proliferation, differentiation or inhibition and/ordeath in B-cell populations and their precursors. The activity of TR21polypeptides, or agonists or antagonists thereof on purified humantonsillar B cells, measured qualitatively over the dose range from 0.1to 10,000 ng/ml, is assessed in a standard B-lymphocyte co-stimulationassay in which purified tonsillar B cells are cultured in the presenceof either formalin-fixed Staphylococcus aureus Cowan I (SAC) orimmobilized anti-human IgM antibody as the priming agent. Second signalssuch as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicitB cell proliferation as measured by tritiated-thymidine incorporation.Novel synergizing agents can be readily identified using this assay. Theassay involves isolating human tonsillar B cells by magnetic bead (MACS)depletion of CD3-positive cells. The resulting cell population isgreater than 95% B cells as assessed by expression of CD45R(B220).Various dilutions of each sample are placed into individual wells of a96-well plate to which are added 10⁵ B-cells suspended in culture medium(RPMI 1640 containing 10% FBS, 5×10⁻⁵M βME, 100 U/ml penicillin, 10ug/ml streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of150u1. Proliferation or inhibition is quantitated by a 20 h pulse(luCi/well) with ³H-thymidine (6.7 Ci/mM) beginning 72 h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

In Vivo Assay

BALB/c mice are injected (i.p.) twice per day with buffer only, or 2mg/Kg of TR21 polypeptide (e.g., soluble TR21) or agonists orantagonists thereof. Mice receive this treatment for 4 consecutive days,at which time they are sacrificed and various tissues and serumcollected for analyses. Comparison of H&E sections from normal and TR21polypeptide-treated spleens identify the results of the activity of TR21polypeptide on spleen cells, such as the diffusion of peri-arteriallymphatic sheaths, and/or significant increases in the nucleatedcellularity of the red pulp regions, which may indicate the activationof the differentiation and proliferation of B-cell populations.Immunohistochemical studies using a B cell marker, anti-CD45R(B220), areused to determine whether any physiological changes to splenic cells,such as splenic disorganization, are due to increased B-cellrepresentation within loosely defined B-cell zones that infiltrateestablished T-cell regions.

Flow cytometric analyses of the spleens from TR21 polypeptide-treatedmice is used to indicate whether TR21 polypeptide specifically increasesthe proportion of ThB+, CD45R(B220) dull B cells over that which isobserved in control mice.

Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andTR21 polypeptide-treated mice.

The studies described in this example test the activity in TR21polypeptide. However, one skilled in the art could easily modify theexemplified studies to test the activity of TR21 polynucleotides (e.g.,gene therapy), and agonists, and/or antagonists of TR21.

Example 14 Assay for TR21 Polypeptide Inhibition of B Cell Proliferationin an In Vitro Co-Stimulatory Assay

This example provides a co-stimulatory assay using Staphylococcus AureusCowan 1 (SAC) as priming agent and Neutrokine-alpha (InternationalApplication Publication No. WO 98/18921) or IL-2 as a second signal toassay for TR21 polypeptide antagonists of Neutrokine-alpha (or IL-2)mediated B cell proliferation.

A soluble TR21 polypeptide is prepared (e.g., a soluble form of TR21corresponding to a portion of the TR21 extracellular domain linked tothe Fc portion of a human IgG1 immunoglobulin molecule). The ability ofthis protein to alter the proliferative response of human B cells isassessed in a standard co-stimulatory assay. Briefly, human tonsillar Bcells are purified by magnetic bead (MACS) depletion of CD3-positivecells. The resulting cell population is routinely greater than 95% Bcells as assessed by expression of CD19 and CD20 staining. Variousdilutions of rHuNeutrokine-alpha (International Application PublicationNo. WO 98/18921) or rHuIL2 are placed into individual wells of a 96-wellplate to which is added 10⁵ B cells suspended in culture medium (RPMI1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100 U/ml penicillin, 10 ug/mlstreptomycin, and 10⁻⁵ dilution of formalin-fixed Staphylococcus aureusCowan I (SAC) also known as Pansorbin (Pan)) in a total volume of 150ul. The TR21 polypeptide is then added at various concentrations and theplates are placed in the incubator (37° C. 5% CO₂, 95% humidity) forthree days. Proliferation is quantitated by a 20 h pulse (1 μCi/well) of³H-thymidine (6.7 Ci/mM) beginning 72 h post factor addition. Thepositive and negative controls are SAC exposed B cells withrHuNeutrokine-alpha (or rHuIL2) and medium (in the absence of the TR21polypeptide), respectively.

Antagonists of rHuNeutrokine-alpha (or rHuIL2) mediated B cellproliferation demonstrate a reduced level of B cell proliferation in thesamples containing the TR21 polypeptides when compared to the positivecontrol.

Example 15 T Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measuredby the uptake of ³H-thymidine. The assay is performed as follows.Ninety-six well plates are coated with 100 μl/well of mAb to CD3 (HIT3a,Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4° C. (1μg/ml in 0.05M bicarbonate buffer, pH 9.5), then washed three times withPBS. PBMC are isolated by F/H gradient centrifugation from humanperipheral blood and added to quadruplicate wells (5×10⁴/well) of mAbcoated plates in RPMI containing 10% FCS and P/S in the presence ofvarying concentrations of TR21 protein (total volume 200 μl). Relevantprotein buffer and medium alone are controls. After 48 hr. culture at37° C., plates are spun for 2 min. at 1000 rpm and 100 μl of supernatantis removed and stored −20° C. for measurement of IL-2 (or othercytokines) if effect on proliferation is observed. Wells aresupplemented with 100 μl of medium containing 0.5 μiCi of ³H-thymidineand cultured at 37° C. for 18-24 hr. Wells are harvested andincorporation of ³H-thymidine used as a measure of proliferation.Anti-CD3 alone is the positive control for proliferation. IL-2 (100U/ml) is also used as a control which enhances proliferation. Controlantibody which does not induce proliferation of T cells is used as thenegative controls for the effects of TR21 proteins.

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 16 Effect of TR21 on the Expression of MHC Class II,Costimulatory and Adhesion Molecules and Cell Differentiation ofMonocytes and Monocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-α,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFCγRII, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

FACS analysis of surface antigens is performed as follows. Cells aretreated 1-3 days with increasing concentrations of TR21 or LPS (positivecontrol), washed with PBS containing 1% BSA and 0.02 mM sodium azide,and then incubated with 1:20 dilution of appropriate FITC- or PE-labeledmonoclonal antibodies for 30 minutes at 4° C. After an additional wash,the labeled cells are analyzed by flow cytometry on a FACScan (BectonDickinson).

Effect on the production of cytokines. Cytokines generated by dendriticcells, in particular IL-12, are important in the initiation of T-celldependent immune responses. IL-12 strongly influences the development ofTh1 helper T-cell immune response, and induces cytotoxic T and NK cellfunction. An ELISA is used to measure the IL-12 release as follows.Dendritic cells (10⁶/ml) are treated with increasing concentrations ofTR21 for 24 hours. LPS (100 ng/ml) is added to the cell culture aspositive control. Supernatants from the cell cultures are then collectedand analyzed for IL-12 content using commercial ELISA kit (e.g., R & DSystems (Minneapolis, Minn.)). The standard protocols provided with thekits are used.

Effect on the expression of MHC Class II, costimulatory and adhesionmolecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inantigen presentation, and Fc receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increase expressionof Fc receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations of TR21 orLPS (positive control), washed with PBS containing 1% BSA and 0.02 mMsodium azide, and then incubated with 1:20 dilution of appropriate FITC-or PE-labeled monoclonal antibodies for 30 minutes at 4° C. After anadditional wash, the labeled cells are analyzed by flow cytometry on aFACScan (Becton Dickinson).

Monocyte activation and/or increased survival. Assays for molecules thatactivate (or alternatively, inactivate) monocytes and/or increasemonocyte survival (or alternatively, decrease monocyte survival) areknown in the art and may routinely be applied to determine whether amolecule of the invention functions as an inhibitor or activator ofmonocytes. TR21, agonists, or antagonists of TR21 can be screened usingthe three assays described below. For each of these assays, Peripheralblood mononuclear cells (PBMC) are purified from single donor leukopacks(American Red Cross, Baltimore, MD) by centrifugation through aHISTOPAQUE™ gradient (Sigma). Monocytes are isolated from PBMC bycounterfiow centrifugal elutriation.

Monocyte Survival Assay. Human peripheral blood monocytes progressivelylose viability when cultured in absence of serum or other stimuli. Theirdeath results from internally regulated process (apoptosis). Addition tothe culture of activating factors, such as TNF-alpha dramaticallyimproves cell survival and prevents DNA fragmentation. Propidium iodide(PI) staining is used to measure apoptosis as follows. Monocytes arecultured for 48 hours in polypropylene tubes in serum-free medium(positive control), in the presence of 100 ng/ml TNF-alpha (negativecontrol), and in the presence of varying concentrations of the compoundto be tested. Cells are suspended at a concentration of 2×10⁶/ml in PBScontaining PI at a final concentration of 5 ng/ml, and then incubated atroom temperature for 5 minutes before FAC Scan analysis. PI uptake hasbeen demonstrated to correlate with DNA fragmentation in thisexperimental paradigm.

Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×10⁵ cells/mlwith increasing concentrations of TR21 and under the same conditions,but in the absence of TR21. For IL-12 production, the cells are primedovernight with IFN-γ (100 U/ml) in presence of TR21. LPS (10 ng/ml) isthen added. Conditioned media are collected after 24 h and kept frozenuntil use. Measurement of TNF-α, IL-10, MCP-1 and IL-8 is then performedusing a commercially available ELISA kit (e.g., R & D Systems(Minneapolis, Minn.)) applying the standard protocols provided with thekit.

Oxidative burst. Purified monocytes are plated in 96-well plate at2-1×10⁵ cell/well. Increasing concentrations of TR21 are added to thewells in a total volume of 0.2 ml culture medium (RPMI 1640+10% FCS,glutamine and antibiotics). After 3 days incubation, the plates arecentrifuged and the medium is removed from the wells. To the macrophagemonolayers, 0.2 ml per well of phenol red solution (140 mM NaCl, 10 mMpotassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol redand 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA).The plates are incubated at 37° C. for 2 hours and the reaction isstopped by adding 20 μl 1N NaOH per well. The absorbance is read at 610nm. To calculate the amount of H₂O₂ produced by the macrophages, astandard curve of a H₂O₂ solution of known molarity is performed foreach experiment.

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 17 The Effect of TR21 on the Growth of Vascular EndothelialCells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at2-5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetalbovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelialcell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the mediumis replaced with M199 containing 10% FBS, 8 units/ml heparin. TR21protein of SEQ ID NO. 2, and positive controls, such as VEGF and basicFGF (bFGF) are added, at varying concentrations. On days 4 and 6, themedium is replaced. On day 8, cell number is determined with a CoulterCounter. An increase in the number of HUVEC cells indicates that TR21may proliferate vascular endothelial cells.

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 18 Stimulatory Effect of TR21 on the Proliferation of VascularEndothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetricMTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)₂H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, PROMEGA™ ). Cells are seeded in a96-well plate (5,000 cells/well) in 0.1 ml serum-supplemented medium andare allowed to attach overnight. After serum-starvation for 12 hours in0.5% FBS, conditions (bFGF, VEGF₁₆₅ or TR21 in 0.5% FBS) with or withoutHeparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMSmixture (1:0.05) are added per well and allowed to incubate for 1 hourat 37° C. before measuring the absorbance at 490 nm in an ELISA platereader. Background absorbance from control wells (some media, no cells)is subtracted, and seven wells are performed in parallel for eachcondition. See, Leak et al. In Vitro Cell. Dev. Biol. 30A:512-518(1994).

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 19 Inhibition of PDGF-Induced Vascular Smooth Muscle CellProliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUincorporation. Briefly, subconfluent, quiescent cells grown on the4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then,the cells are pulsed with 10% calf serum and 6 mg/ml BrdU. After 24 h,immunocytochemistry is performed using the BrdU Staining Kit (ZymedLaboratories). In brief, the cells are incubated with the biotinylatedmouse anti-BrdU antibody at 4° C. for 2 h after exposing to denaturingsolution and then with the streptavidin-peroxidase and diaminobenzidine.After counterstaining with hematoxylin, the cells are mounted formicroscopic examination, and the Positive-positive cells are counted.The Positive index is calculated as a percent of the Positive-positivecells to the total cell number. In addition, the simultaneous detectionof the Positive staining (nucleus) and the FITC uptake (cytoplasm) isperformed for individual cells by the concomitant use of bright fieldillumination and dark field-UV fluorescent illumination. See, Hayashidaet al., J. Biol. Chem. 6;271(36):21985-21992 (1996).

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 20 Stimulation of Endothelial Migration

This example will be used to explore the possibility that TR21 maystimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48 wellmicrochemotaxis chamber (Neuroprobe Inc., Cabin John, M D; Falk, W.,Goodwin, R. H. J., and Leonard, E. J. “A 48 well micro chemotaxisassembly for rapid and accurate measurement of leukocyte migration.” J.Immunological Methods 1980; 33:239-247). Polyvinylpyrrolidone-freepolycarbonate filters with a pore size of 8 um (Nucleopore Corp.Cambridge, Mass.) are coated with 0.1% gelatin for at least 6 hours atroom temperature and dried under sterile air. Test substances arediluted to appropriate concentrations in M199 supplemented with 0.25%bovine serum albumin (BSA), and 25 ul of the final dilution is placed inthe lower chamber of the modified Boyden apparatus. Subconfluent, earlypassage (2-6) HUVEC or BMEC cultures are washed and trypsinized for theminimum time required to achieve cell detachment. After placing thefilter between lower and upper chamber, 2.5×10⁵ cells suspended in 50 ulM199 containing 1% FBS are seeded in the upper compartment. Theapparatus is then incubated for 5 hours at 37° C. in a humidifiedchamber with 5% CO2 to allow cell migration. After the incubationperiod, the filter is removed and the upper side of the filter with thenon-migrated cells is scraped with a rubber policeman. The filters arefixed with methanol and stained with a Giemsa solution (Diff-Quick,Baxter, McGraw Park, Ill.). Migration is quantified by counting cells ofthree random high-power fields (40×) in each well, and all groups areperformed in quadruplicate.

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 21 Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be amediator of vascular endothelium relaxation. Thus, TR21 activity can beassayed by determining nitric oxide production by endothelial cells inresponse to TR21.

Nitric oxide is measured in 96-well plates of confluent microvascularendothelial cells after 24 hours starvation and a subsequent 4 hrexposure to various levels of a positive control (such as VEGF-1) andTR21. Nitric oxide in the medium is determined by use of the Griessreagent to measure total nitrite after reduction of nitric oxide-derivednitrate, by nitrate reductase. The effect of TR21 on nitric oxiderelease is examined on HUVEC.

Briefly, NO release from cultured HUVEC monolayer is measured with aNO-specific polarographic electrode connected to a NO meter (Iso-NO,World Precision Instruments Inc.). Calibration of the NO element isperformed according to the following equation:

2KNO₂+2KI+2H₂SO₄ 6 2NO+I₂+2H₂O+2K₂SO₄

The standard calibration curve is obtained by adding gradedconcentrations of KNO₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) intothe calibration solution containing KI and H₂SO₄. The specificity of theIso-NO electrode to NO is previously determined by measurement of NOfrom authentic NO gas. The culture medium is removed and HUVECs arewashed twice with Dulbecco's phosphate buffered saline. The cells arethen bathed in 5 ml of filtered Krebs-Henseleit solution in 6-wellplates, and the cell plates are kept on a slide warmer (Lab LineInstruments Inc.) to maintain the temperature at 37° C. The NO sensorprobe is inserted vertically into the wells, keeping the tip of theelectrode 2 mm under the surface of the solution, before addition of thedifferent conditions. S-nitroso acetyl penicillamin (SNAP) is used as apositive control. The amount of released NO is expressed as picomolesper 1×10⁶ endothelial cells. All values reported are means of four tosix measurements in each group (number of cell culture wells). See, Leaket al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 22 Effect of TR21 on Cord Formation in Angiogenesis

Another step in angiogenesis is cord formation, marked bydifferentiation of endothelial cells. This bioassay measures the abilityof microvascular endothelial cells to form capillary-like structures(hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) are purchased from CellApplications, Inc. as proliferating (passage 2) cells and are culturedin Cell Applications' CADMEC Growth Medium and used at passage 5. Forthe in vitro angiogenesis assay, the wells of a 48-well cell cultureplate are coated with Cell Applications' Attachment Factor Medium (200μl/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wellsat 7,500 cells/well and cultured overnight in Growth Medium. The GrowthMedium is then replaced with 300 μg Cell Applications' Chord FormationMedium containing control buffer or TR21 (0.1 to 100 ng/ml) and thecells are cultured for an additional 48 hr. The numbers and lengths ofthe capillary-like chords are quantitated through use of the BoeckelerVIA-170 video image analyzer. All assays are done in triplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.β-esteradiol (1 ng/ml) is used as a negative control. The appropriatebuffer (without protein) is also utilized as a control.

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 23 Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system toexamine angiogenesis. Blood vessel formation on CAM is easily visibleand quantifiable. The ability of TR21 to stimulate angiogenesis in CAMcan be examined.

Fertilized eggs of the White Leghorn chick (Gallus gallus) and theJapanese quail (Coturnix coturnix) are incubated at 37.8° C. and 80%humidity. Differentiated CAM of 16-day-old chick and 13-day-old quailembryos is studied with the following methods.

On Day 4 of development, a window is made into the eggshell of chickeggs. The embryos are checked for normal development and the eggs sealedwith cellotape. They are further incubated until Day 13. THERMANOX™coverslips (Nunc, Naperville, Ill.) are cut into disks of about 5 mm indiameter. Sterile and salt-free growth factors, and the protein to betested, are dissolved in distilled water and about 3.3 mg/5 ml arepipetted on the disks. After air-drying, the inverted disks are appliedon CAM. After 3 days, the specimens are fixed in 3% glutaraldehyde and2% formaldehyde and rinsed in 0.12 M sodium cacodylate buffer. They arephotographed with a stereo microscope [Wild M8] and embedded for semi-and ultrathin sectioning as described above. Controls are performed withcarrier disks alone.

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 24 Angiogenesis Assay Using a MATRIGEL™ Implant in Mouse

In order to establish an in vivo model for angiogenesis to test TR21protein activities, mice and rats are implanted subcutaneously withmethylcellulose disks containing either 20 mg of BSA (negative control),1 mg of TR21, or 0.5 mg of VEGF-1 (positive control). The negativecontrol disks should contain little vascularization, while the positivecontrol disks should show signs of vessel formation.

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 25 Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of TR21 on ischemia, a rabbit hindlimbischemia model is created by surgical removal of one femoral arteries asdescribed previously (Takeshita, S. et al., Am J. Pathol 147:1649-1660(1995)). The excision of the femoral artery results in retrogradepropagation of thrombus and occlusion of the external iliac artery.Consequently, blood flow to the ischemic limb is dependent uponcollateral vessels originating from the internal iliac artery(Takeshita, S. et al., Am J. Pathol 147:1649-1660 (1995)). An intervalof 10 days is allowed for post-operative recovery of rabbits anddevelopment of endogenous collateral vessels. At 10 day post-operatively(day 0), after performing a baseline angiogram, the internal iliacartery of the ischemic limb is transfected with 500 mg naked TR21expression plasmid by arterial gene transfer technology using ahydrogel-coated balloon catheter as described (Riessen, R. et al., HumGene Ther. 4:749-758 (1993); Leclerc, G. et al., J. Clin. Invest. 90:936-944 (1992)). When TR21 is used in the treatment, a single bolus of500 mg TR21 protein or control is delivered into the internal iliacartery of the ischemic limb over a period of 1 min. through an infusioncatheter. On day 30, various parameters are measured in these rabbits:(a) BP ratio—The blood pressure ratio of systolic pressure of theischemic limb to that of normal limb; (b) Blood Flow and FlowReserve—Resting FL: the blood flow during undilated condition and MaxFL: the blood flow during fully dilated condition (also an indirectmeasure of the blood vessel amount) and Flow Reserve is reflected by theratio of max FL: resting FL; (c) Angiographic Score—This is measured bythe angiogram of collateral vessels. A score is determined by thepercentage of circles in an overlaying grid that with crossing opacifiedarteries divided by the total number m the rabbit thigh; (d) Capillarydensity—The number of collateral capillaries determined in lightmicroscopic sections taken from hindlimbs.

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 26 Rat Ischemic Skin Flap Model

The evaluation parameters include skin blood flow, skin temperature, andfactor VIII immunohistochemistry or endothelial alkaline phosphatasereaction. TR21 expression, during the skin ischemia, is studied using insitu hybridization.

The study in this model is divided into three parts as follows:

-   -   Ischemic skin    -   Ischemic skin wounds    -   Normal wounds

The experimental protocol includes:

-   -   Raising a 3×4 cm, single pedicle full-thickness random skin flap        (myocutaneous flap over the lower back of the animal).    -   An excisional wounding (4-6 mm in diameter) in the ischemic skin        (skin-flap).    -   Topical treatment with TR21 of the excisional wounds (day 0, 1,        2, 3, 4 post-wounding) at the following various dosage ranges: 1        mg to 100 mg.    -   Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21        post-wounding for histological, immunohistochemical, and in situ        studies.

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 27 Peripheral Arterial Disease Model

Angiogenic therapy using TR21 is a novel therapeutic strategy to obtainrestoration of blood flow around the ischemia in case of peripheralarterial diseases. The experimental protocol includes:

a) One side of the femoral artery is ligated to create ischemic muscleof the hindlimb, the other side of hindlimb serves as a control.

b) TR21 protein, in a dosage range of 20 mg -500 mg, is deliveredintravenously and/or intramuscularly 3 times (perhaps more) per week for2-3 weeks.

c) The ischemic muscle tissue is collected after ligation of the femoralartery at 1, 2, and 3 weeks for the analysis of TR21 expression andhistology. Biopsy is also performed on the other side of normal muscleof the contralateral hindlimb

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 28 Ischemic Myocardial Disease Model

TR21 is evaluated as a potent mitogen capable of stimulating thedevelopment of collateral vessels, and restructuring new vessels aftercoronary artery occlusion. Alteration of TR21 expression is investigatedin situ. The experimental protocol includes:

a) The heart is exposed through a left-side thoracotomy in the ratImmediately, the left coronary artery is occluded with a thin suture(6-0) and the thorax is closed.

b) TR21 protein, in a dosage range of 20 mg -500 mg, is deliveredintravenously and/or intramuscularly 3 times (perhaps more) per week for2-4 weeks.

c) Thirty days after the surgery, the heart is removed andcross-sectioned for morphometric and in situ analyzes.

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 29 Rat Corneal Wound Healing Model

This animal model shows the effect of TR21 on neovascularization. Theexperimental protocol includes:

a) Making a 1-1.5 mm long incision from the center of cornea into thestromal layer.

b) Inserting a spatula below the lip of the incision facing the outercorner of the eye.

c) Making a pocket (its base is 1-1.5 mm form the edge of the eye).

d) Positioning a pellet, containing 50 ng-5 ug of TR21, within thepocket.

e) TR21 treatment can also be applied topically to the corneal wounds ina dosage range of 20 mg-500 mg (daily treatment for five days).

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 30 Diabetic Mouse and Glucocorticoid-Impaired Wound HealingModels

A. Diabetic db+/db+ Mouse Model.

To demonstrate that TR21 accelerates the healing process, thegenetically diabetic mouse model of wound healing is used. The fullthickness wound healing model in the db+/db+ mouse is a wellcharacterized, clinically relevant and reproducible model of impairedwound healing. Healing of the diabetic wound is dependent on formationof granulation tissue and re-epithelialization rather than contraction(Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhaigh, D. G.et al., Am. J. Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293(1982)). Animals show polyphagia, polydipsia and polyuria. Mutantdiabetic mice (db+/db+) have elevated blood glucose, increased or normalinsulin levels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979);Coleman, D. L., Diabetes 31 (Suppl): 1-6 (1982)). These homozygousdiabetic mice develop hyperglycemia that is resistant to insulinanalogous to human type II diabetes (Mandel et al., J. Immunol.120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andwere 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals

Wounding protocol is performed according to previously reported methods(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of surgery and at two day intervals thereafter. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

TR21 is administered using at a range different doses of TR21, from 4 mgto 500 mg per wound per day for 8 days in vehicle. Vehicle controlgroups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)are evaluated: 1) Vehicle placebo control, 2) TR21.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 was 64 mm², the corresponding size of the dermalpunch. Calculations were made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with TR21. This assessment included verification of thepresence of cell accumulation, inflammatory cells, capillaries,fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D.G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometeris used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin is used as a positive tissue control while non-immune IgG isused as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens isdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer served as a positive tissue controland human brain tissue is used as a negative tissue control. Eachspecimen included a section with omission of the primary antibody andsubstitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0-8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

B. Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, S. M. Glucocorticoids andWound healing. In: Anti-Inflammatory Steroid Action: Basic and ClinicalAspects. 280-302 (1989); Wahl, S. M. et al., J. Immunol. 115: 476-481(1975); Werb, Z. et al., J. Exp. Med. 147:1684-1694 (1978)).Glucocorticoids retard wound healing by inhibiting angiogenesis,decreasing vascular permeability (Ebert, R. H., et al., An. Intern. Med.37:701-705 (1952)), fibroblast proliferation, and collagen synthesis(Beck, L. S. et al., Growth Factors. 5: 295-304 (1991); Haynes, B. F. etal., J. Clin. Invest. 61: 703-797 (1978)) and producing a transientreduction of circulating monocytes (Haynes, B. F., et al., J. Clin.Invest. 61: 703-797 (1978); Wahl, S. M., “Glucocorticoids and woundhealing”, In: Anti-inflammatory Steroid Action: Basic and ClinicalAspects, Academic Press, New York, pp. 280-302 (1989)). The systemicadministration of steroids to impaired wound healing is a well establishphenomenon in rats (Beck, L. S. et al., Growth Factors. 5: 295-304(1991); Haynes, B. F., et al., J. Clin. Invest. 61: 703-797 (1978);Wahl, S. M., “Glucocorticoids and wound healing”, In: Anti-inflammatorySteroid Action: Basic and Clinical Aspects, Academic Press, New York,pp. 280-302 (1989); Pierce, G. F. et al., Proc. Natl. Acad. Sci. USA 86:2229-2233 (1989)).

To demonstrate that TR21 can accelerate the healing process, the effectsof multiple topical applications of TR21 on full thickness excisionalskin wounds in rats in which healing has been impaired by the systemicadministration of methylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles RiverLaboratories) are used in this example. The animals are purchased at 8weeks of age and were 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy is conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to section A, above. On theday of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of wounding and at the end of treatment. Wound closure is determinedby daily measurement on days 1-5 and on day 8. Wounds are measuredhorizontally and vertically using a calibrated Jameson caliper. Woundsare considered healed if granulation tissue was no longer visible andthe wound is covered by a continuous epithelium.

TR21 is administered using at a range different doses of TR21, from 4 mgto 500 mg per wound per day for 8 days in vehicle. Vehicle controlgroups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

Four groups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) were evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) TR21 treated groups.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total area of the wound. Closure isthen estimated by establishing the differences between the initial woundarea (day 0) and that of post treatment (day 8). The wound area on day 1was 64mm², the corresponding size of the dermal punch. Calculations weremade using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5mm) and cutusing an Olympus microtome. Routine hematoxylin-eosin (H&E) staining wasperformed on cross-sections of bisected wounds. Histologic examinationof the wounds allows assessment of whether the healing process and themorphologic appearance of the repaired skin was improved by treatmentwith TR21. A calibrated lens micrometer is used by a blinded observer todetermine the distance of the wound gap.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

Example 31 Lymphedema Animal Model

The purpose of this experimental approach is to create an appropriateand consistent lymphedema model for testing the therapeutic effects ofTR21 in lymphangiogenesis and re-establishment of the lymphaticcirculatory system in the rat hind limb. Effectiveness is measured byswelling volume of the affected limb, quantification of the amount oflymphatic vasculature, total blood plasma protein, and histopathology.Acute lymphedema is observed for 7-10 days. Perhaps more importantly,the chronic progress of the edema is followed for up to 3-4 weeks.

Prior to beginning surgery, blood sample is drawn for proteinconcentration analysis. Male rats weighing approximately ˜350 g aredosed with Pentobarbital. Subsequently, the right legs are shaved fromknee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH.Blood is drawn for serum total protein testing. Circumference andvolumetric measurements are made prior to injecting dye into paws aftermarking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsalpaw). The intradermal dorsum of both right and left paws are injectedwith 0.05 ml of 1% Evan's Blue. Circumference and volumetricmeasurements are then made following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is madecircumferentially allowing the femoral vessels to be located. Forcepsand hemostats are used to dissect and separate the skin flaps. Afterlocating the femoral vessels, the lymphatic vessel that runs along sideand underneath the vessel(s) is located. The main lymphatic vessels inthis area are then electrically coagulated or suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosisand adductors) are bluntly dissected. The popliteal lymph node is thenlocated.

The 2 proximal and 2 distal lymphatic vessels and distal blood supply ofthe popliteal node are then and ligated by suturing. The popliteal lymphnode, and any accompanying adipose tissue, is then removed by cuttingconnective tissues.

Care is taken to control any mild bleeding resulting from thisprocedure. After lymphatics are occluded, the skin flaps are sealed byusing liquid skin (Vetbond) (AJ Buck). The separated skin edges aresealed to the underlying muscle tissue while leaving a gap of ˜0.5 cmaround the leg. Skin also may be anchored by suturing to underlyingmuscle when necessary.

To avoid infection, animals are housed individually with mesh (nobedding). Recovering animals are checked daily through the optimaledematous peak, which typically occurred by day 5-7. The plateauedematous peak are then observed. To evaluate the intensity of thelymphedema, the circumference and volumes of 2 designated places on eachpaw before operation and daily for 7 days are measured. The effectplasma proteins on lymphedema is determined and whether protein analysisis a useful testing perimeter is also investigated. The weights of bothcontrol and edematous limbs are evaluated at 2 places. Analysis isperformed in a blind manner.

Circumference Measurements: Under brief gas anesthetic to prevent limbmovement, a cloth tape is used to measure limb circumference.Measurements are done at the ankle bone and dorsal paw by 2 differentpeople then those 2 readings are averaged. Readings are taken from bothcontrol and edematous limbs.

Volumetric Measurements: On the day of surgery, animals are anesthetizedwith Pentobarbital and are tested prior to surgery. For dailyvolumetrics animals are under brief halothane anesthetic (rapidimmobilization and quick recovery), both legs are shaved and equallymarked using waterproof marker on legs. Legs are first dipped in water,then dipped into instrument to each marked level then measured by Buxcoedema software (Chen/Victor). Data is recorded by one person, while theother is dipping the limb to marked area.

Blood-plasma protein measurements: Blood is drawn, spun, and serumseparated prior to surgery and then at conclusion for total protein andCa2+ comparison.

Limb Weight Comparison: After drawing blood, the animal is prepared fortissue collection. The limbs were amputated using a guillotine, thenboth experimental and control legs were cut at the ligature and weighed.A second weighing is done as the tibio-cacaneal joint was disarticulatedand the foot was weighed.

Histological Preparations: The transverse muscle located behind the knee(popliteal) area is dissected and arranged in a metal mold, filled withfreezeGel, dipped into cold methylbutane, placed into labeled samplebags at −80EC until sectioning. Upon sectioning, the muscle was observedunder fluorescent microscopy for lymphatics. Other immuno/histologicalmethods are currently being evaluated.

The studies described in this example test the activity in TR21 protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of TR21 polynucleotides (e.g., genetherapy), agonists, and/or antagonists of TR21.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, and Examples is hereby incorporated herein by reference.

Further, the Sequence Listing submitted herewith, in both computer andpaper forms, is hereby incorporated by reference in its entirety.

Moreover, the disclosure of U.S. Provisional Patent Application Ser. No.60/315,357 is hereby incorporated by reference in its entirety.

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence at least 90% identical to a sequence selected from the groupconsisting of: (a) amino acids from 2 to 81 in SEQ ID NO:2 (FIG. 1); (b)amino acids from 2 to 38 in SEQ ID NO:2 (FIG. 1); (c) amino acids from 2to 46 in SEQ ID NO:2 (FIG. 1); (d) amino acids from 2 to 62 in SEQ IDNO:2 (FIG. 1); and optionally, a heterologous polypeptide sequence
 2. Anisolated nucleic acid molecule encoding the polypeptide of claim
 1. 3. Arecombinant vector or a recombinant host cell comprising nucleic acidmolecule of claim
 2. 4. An isolated antibody that binds specifically toa polypeptide consisting of amino acid residues selected from the groupconsisting of: (a) amino acids 2 to 81 in SEQ ID NO:2; (b) amino acids 2to 38 in SEQ ID NO:2; (c) amino acids 2 to 46 in SEQ ID NO:2; (d) aminoacids 2 to 62 in SEQ ID NO:2; (e) amino acids 3 to 15 in SEQ ID NO:2;(f) amino acids 39 to 45 in SEQ ID NO:2; (g) amino acids 51 to 53 in SEQID NO:2; (h) amino acids 59 to 61 in SEQ ID NO:2; (i) amino acids 104 to112 in SEQ ID NO:2; (j) amino acids 115 to 122 in SEQ ID NO:2; (k) aminoacids 136 to 138 in SEQ ID NO:2; (l) amino acids 147 to 153 in SEQ IDNO:2; and (m) amino acids 178 to 182 in SEQ ID NO:2.
 5. The antibody ofclaim 4 which binds a polypeptide consisting of amino acids 2 to 38 ofSEQ ID NO:2 and a polypeptide consisting of amino acids 2 to 81 of SEQID NO:2.
 6. A method of treating an autoimmune disease or conditionassociated with an autoimmune disease, comprising administering aneffective amount of the polypeptide of claim 1, to a patient in needthereof; wherein said autoimmune disease is selected from the groupconsisting of: (a) rheumatoid arthritis; (b) systemic lupuserythmatosus; (c) multiple sclerosis; (d) Sjogren's syndome; (e) IgAnephropathy; (f) glomerulonephritis; (g) diabetes mellitus; and (h)myasthenia gravis.
 7. A method of treating an autoimmune disease orcondition associated with an autoimmune disease comprising,administering an effective amount of the antibody of claim 4, to apatient in need thereof; wherein said autoimmune disease is selectedfrom the group consisting of: (a) rheumatoid arthritis; (b) systemiclupus erythmatosus; (c) multiple sclerosis; (d) Sjogren's syndome; (e)IgA nephropathy; (¹) glomerulonephritis; (g) diabetes mellitus; and (h)myasthenia gravis.
 8. A method of treating an immunodeficiency orcondition associated with an immunodeficiency, comprising administeringan effective amount of the antibody of claim 4, to a patient in needthereof; wherein said immunodeficiency is selected from the groupconsisting of: (a) common variable immunodeficiency (CVID); (b) acquiredimmunodeficiency syndrome (AIDS); (c) severe combined immunodeficiency(SCID); (d) selective IgA deficiency; (e) hypogammaglobulinemia; and (f)Wiskott-Aldrich syndrome.
 9. A method of diagnosing an immunodeficiencyor an autoimmune disease, comprising contacting the polypeptide of claim1 with cells or bodily fluids from an individual, and assaying forbinding to said polypeptide.
 10. A method of diagnosing animmunodeficiency or an autoimmune disease, comprising contacting theantibody of claim 4 with cells or bodily fluids from an individual, andassaying for binding to said antibody.
 11. A method of increasing B cellproliferation or immunoglobulin production, comprising administering aneffective amount of the antibody of claim 4, to a patient in needthereof.
 12. A method of inhibiting B cell proliferation orimmunoglobulin production, comprising administering an effective amountof the polypeptide of claim 1 to a patient in need thereof.
 13. A methodof inhibiting B cell proliferation or immunoglobulin production,comprising administering an effective amount of the antibody of claim 4to a patient in need thereof.
 14. A method of killing a cell thatexpresses TR21 polypeptide on its cell surface, comprising contactingsaid cell with an antibody or portion thereof that specifically binds apolypeptide consisting of amino acid residues 1-184 of SEQ ID NO:2;wherein said antibody or portion thereof is conjugated to a toxin. 15.The method of claim 14 performed in vivo.
 16. The method of claim 14wherein said antibody or portion thereof specifically binds to apolypeptide consisting of amino acid residues 1-81 of SEQ ID NO:2. 17.The method of claim 14 wherein the antibody or portion thereof ismonoclonal, chimeric or human.
 18. The method of claim 14 wherein theantibody or portion thereof is a single chain antibody or a Fabfragment.
 19. The method of claim 14 wherein said cell is a lymphocyte.20. The method of claim 19 wherein said lymphocyte is leukemic.